Pathogens Penetrating the Central Nervous System: InfectionPathways and the Cellular and Molecular Mechanisms of InvasionSamantha J. Dando,a Alan Mackay-Sim,b Robert Norton,c Bart J. Currie,d James A. St. John,b Jenny A. K. Ekberg,b,e Michael Batzloff,a

Glen C. Ulett,f Ifor R. Beachama

Institute for Glycomics, Griffith University, Gold Coast, Queensland, Australiaa; Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australiab;Townsville Hospital, Townsville, Queensland, Australiac; Menzies School of Health Research and Royal Darwin Hospital, Darwin, Northern Territory, Australiad; School ofBiomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australiae; School of Medical Science and Griffith Health Institute, Griffith University,Gold Coast, Queensland, Australiaf

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .692INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .692

Meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .692Pneumococcal meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .692Meningococcal meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .693H. influenzae, L. monocytogenes, and other bacterial causes of meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .693

Encephalitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .693Focal Infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .694

BARRIERS OF THE CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .694The Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .694The Blood-Cerebrospinal Fluid Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .694Immunosurveillance of the CNS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .695

MICROBIAL ENTRY TO THE CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .696In Vivo and In Vitro Models of CNS Invasion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .696

BACTERIAL INVASION THROUGH THE BLOOD-BRAIN AND BLOOD-CEREBROSPINAL FLUID BARRIERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .697Sites of Entry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .697Mechanisms of Entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .697Transcellular Penetration of Brain Microvascular Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .699

E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .699N. meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .700S. pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .700Group B streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .701S. suis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .701L. monocytogenes and B. pseudomallei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .702M. tuberculosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .703

Paracellular Penetration of Brain Microvascular Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .703E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .703H. influenzae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .704N. meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .704Streptococcus spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .704

Trojan Horse Penetration of Brain Microvascular Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .705THE OLFACTORY NERVE AS A PORTAL TO THE BRAIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .705THE TRIGEMINAL NERVE AS A PORTAL TO THE BRAIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .707PROTECTING THE CNS FROM MICROBIAL INVASION VIA THE NASAL CAVITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .707PATHOGENS THAT ENTER THE BRAIN THROUGH THE NOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .708

Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .708S. pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .708N. meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .709B. pseudomallei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .709L. monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .710

Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .710Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .712Yeasts and Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .712

(continued)

Address correspondence to Glen C. Ulett, g.ulett@griffith.edu.au, or Ifor R.Beacham, i.beacham@griffith.edu.au.

G.C.U. and I.R.B. contributed equally to this work.

Copyright 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CMR.00118-13

October 2014 Volume 27 Number 4 Clinical Microbiology Reviews p. 691726 cmr.asm.org 691

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://dx.doi.org/10.1128/CMR.00118-13http://cmr.asm.orghttp://cmr.asm.org/

CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .712REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .713AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .725

SUMMARY

The brain is well protected against microbial invasion by cellularbarriers, such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). In addition, cells within thecentral nervous system (CNS) are capable of producing an im-mune response against invading pathogens. Nonetheless, a rangeof pathogenic microbes make their way to the CNS, and the re-sulting infections can cause significant morbidity and mortality.Bacteria, amoebae, fungi, and viruses are capable of CNS invasion,with the latter using axonal transport as a common route of infec-tion. In this review, we compare the mechanisms by which bacterialpathogens reach the CNS and infect the brain. In particular, we focuson recent data regarding mechanisms of bacterial translocation fromthe nasal mucosa to the brain, which represents a little explored path-way of bacterial invasion but has been proposed as being particularlyimportant in explaining how infection with Burkholderia pseu-domallei can result in melioidosis encephalomyelitis.

INTRODUCTION

Microbial infection involving the central nervous system(CNS) is an important and relatively common presentation.CNS infections are frequently caused by viruses, such as the en-teroviruses, which cause the majority of cases of aseptic meningitisand meningoencephalitis (13). Other neurotropic viruses, suchas human cytomegalovirus, herpes simplex viruses, varicella-zostervirus, and the emerging viruses West Nile virus, henipaviruses, Japa-nese encephalitis virus, chikungunya virus, Ebola virus, and rabiesvirus, may also cause CNS infections (47). There are many bacterialpathogens that are associated with CNS invasion. Rapid detection ofa bacterial agent in such presentations and the initiation of appropri-ate antibiotic therapy influence morbidity and mortality (810). Theclinical presentations of bacterial CNS infection range from menin-gitis and meningoencephalitis to focal CNS syndromes.

Meningitis

Meningitis, or inflammation of the meninges, is usually acute butcan also be subacute and most frequently presents with headache,fever, and neck stiffness (11). Meningitis can be pyogenic (pusforming), which is associated with common bacterial etiologies(described below), or aseptic, in which pyogenic bacteria are notisolated from the cerebrospinal fluid (CSF) and the causativeagent may be viral or mycobacterial or there is a noninfectiveetiology. Features of CSF which would generally exclude a bacte-rial etiology for meningitis include a protein concentration of600 mg/liter and a white cell count of 90 106/liter (2). Theclassic triad of fever, neck stiffness, and a change in mental state isobserved in 44% of patients with bacterial meningitis (11).

A prospective, laboratory-based surveillance study in 1986 de-fined the epidemiology of bacterial meningitis in a population of34 million in the United States (12). This study reported an aver-age of 6.7 cases of bacterial meningitis per 100,000 populationacross six states and demonstrated that Haemophilus influenzae,Neisseria meningitidis, and Streptococcus pneumoniae caused 77%

of cases, which was consistent with a previous report by the Na-tional Surveillance of Bacterial Meningitis (13). More recently, itwas reported that the incidence of bacterial meningitis in theUnited States declined by 31% during the surveillance period of1998 to 2007, most likely due to the success of vaccine programs(14). S. pneumoniae was the most common cause of bacterialmeningitis (58.0%), followed by Streptococcus agalactiae (group Bstreptococcus) (18.1%), N. meningitidis (13.9%), H. influenzae(6.7%), and Listeria monocytogenes (3.4%). Staphylococcus aureusand Escherichia coli are also important causes of bacterial menin-gitis (15), and Streptococcus suis is emerging as a zoonotic etiologyof meningitis (16); however, it must be noted that meningitis canbe caused by a plethora of different bacteria (17). The most com-mon alternative bacterial cause of meningitis is Mycobacteriumtuberculosis, which is recognized particularly in pediatric popula-tions of the developing world (18, 19). These infections involve aclinical onset that is usually insidious, with hydrocephalus being aprominent feature.

Bacterial meningitis is usually preceded by nasopharyngeal ormiddle ear colonization, followed by (i) microbial invasion of thetissue and intravascular space and (ii) bacteremia. Meningeal in-vasion occurs following penetration of the cellular barriers of theCNS. High-level bacteremia was shown to be necessary for thedevelopment of meningitis in experimental animal models (20,21), which is consistent with clinical observations in humans (2224). Bacteria that commonly cause meningitis express antiphago-cytic capsular polysaccharide, which enables survival/multiplica-tion within the blood. Several species-specific factors thatpromote bacterial survival in blood, resistance to complement,and survival within phagocytes have been identified (2530). Bac-terial meningitis resulting from nonhematogenous spread is lesscommon and is a consequence of infection preexisting in an adja-cent intracranial site. Such infections include mastoiditis, frontalsinusitis, and rupture of an intracerebral abscess into the ventri-cles of the brain and may occur following head trauma or neuro-surgical procedures.

Pneumococcal meningitis. There are no specific distinguishingclinical features of pneumococcal meningitis. While a rash is un-common, it is occasionally seen in splenectomized patients withoverwhelming sepsis. There is a close correlation between bacte-remic and meningitic serotypes of S. pneumoniae, implicating he-matogenous spread as the commonest mode of acquisition (31). S.pneumoniae is the most frequent cause of bacterial meningitis inthe United States and has a case fatality rate of 14.7% (14). Afterthe introduction of the 7-valent pneumococcal conjugate vaccine(which contains serotype antigens 4, 6B, 9V, 14, 18C, 19F, and23F) in the United States, the incidence of pneumococcal menin-gitis declined by 30.1%, from 1.13 cases per 100,000 population in1998 to 1999 to 0.79 cases per 100,000 population in 2004 to 2005(32). In patients of 2 years and 65 years of age, the incidence ofpneumococcal meningitis throughout the surveillance period de-clined by 64.0% and 54.0%, respectively. Across all age groups, thenumber of meningitis cases caused by S. pneumoniae serotypes

Dando et al.

692 cmr.asm.org Clinical Microbiology Reviews

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

covered by the 7-valent vaccine dropped by 73.3% (32). Similarreductions in invasive pneumococcal disease caused by 7-valentserotypes have also been reported in Australia, England, andWales (3335); however, this has led to a replacement phenome-non, in which the rates of disease caused by non-7-valent S. pneu-moniae serotypes have significantly increased (32, 34, 35).

Meningococcal meningitis. N. meningitidis is most likely tocause meningitis in children and adolescents and has a mortalityrate of 10.1% in the United States (14). The serotypes most com-monly implicated are A, B, C, W135, and Y (14, 3638), and men-ingitis without shock is the most common presentation (38). Thepredominant clinical feature, which can distinguish N. meningiti-dis from other causes of bacterial meningitis, is the presence of apetechial rash. It can rapidly become purpuric, which in the pres-ence of meningitis or sepsis strongly implicates N. meningitidis asthe etiologic agent. These lesions are a consequence of meningo-cocci adhering to the endothelial cells of the capillaries and smallveins in the skin, thereby altering the antithrombotic surface of theendothelium. This results in the formation of clots and the ex-travasion of erythrocytes, which appear as skin hemorrhages. Le-sions of 1.0 cm usually occur in patients developing shock, withhigh levels of circulating endotoxin (38).

Immunization against N. meningitidis serotypes A and C hasresulted in a significant decrease in meningococcal disease. TheUnited Kingdom was the first country to introduce routine im-munization with a monovalent serotype C conjugate vaccine, in1999. During the first follow-up surveillance period (2000 to2001), the incidence of serotype C meningococcal disease fell by80% in target age groups (39). Meningococcal serotype C vacci-nation programs in Australia have also led to a marked decrease indisease due to N. meningitidis serotype C. Of the 90% of menin-gococcal cases in Australia in 2010 that had serotype data avail-able, N. meningitidis serotype B caused 84.0% of cases (40). In2013, a recombinant meningococcus serotype B vaccine (Bexsero)was added to the Australian Register of Therapeutic Goods for usein individuals of 2 months of age and was also approved for usein Europe. In New Zealand, the introduction of an outer mem-brane vesicle vaccine against an epidemic N. meningitidis serotypeB clone in New Zealand was reported to be 80.0% effective in fullyimmunized children aged 6 months to 5 years (41).

H. influenzae, L. monocytogenes, and other bacterial causes ofmeningitis. H. influenzae as a cause of meningitis is now uncom-mon in most Western countries due to widespread immunizationagainst H. influenzae capsular type B, which was previously re-sponsible for the majority of cases of H. influenzae meningitis. Asa cause of meningitis, H. influenzae has fallen from 45.0% in 1986(12) to 6.7% in 2003 to 2007 (14). L. monocytogenes is also aninfrequent cause of meningitis; however, it is associated with highmortality rates (17.0% to 28.0%) (14, 42, 43). L. monocytogenesmeningitis occurs primarily in children of 2 months of age andin immunocompromised and elderly individuals (14, 42, 43). Thesigns and symptoms of L. monocytogenes meningitis are not dif-ferent from those associated with other causes of community-acquired meningitis, but CSF findings may be atypical (43, 44).Listeriosis is associated with the consumption of contaminatedfood, implicating the gastrointestinal tract as the portal of entry tothe bloodstream.

Group B streptococcus and E. coli are common causes of men-ingitis in neonates (45, 46), with group B streptococcus account-ing for 86.1% of bacterial meningitis cases in patients of 2

months of age (14). Gram-negative aerobic bacteria, such as Kleb-siella pneumoniae, Serratia marcescens, Salmonella spp., and Pseu-domonas aeruginosa, can occasionally be responsible for meningi-tis in certain patient groups, including infants and patients withhead injuries or those undergoing postneurosurgical proceduresor immunosuppression (31, 47, 48). M. tuberculosis is a commoncause of chronic or subacute meningitis (49) and is associated withnonspecific symptoms and an absence of neck stiffness duringearly disease (50). Tuberculosis meningitis occurs following he-matogenous spread of bacilli from the lungs and the establishmentof a caseating Rich focus (tuberculous granuloma) in the braincortex, meninges, or choroid plexus. Bacteria are subsequentlyreleased from the foci and enter the subarachnoid space (51).

Encephalitis

In certain circumstances, acute meningitis can be clinically indis-tinguishable from acute encephalitis, which refers to inflamma-tion of the brain parenchyma in association with neurologic dys-function (52). Encephalitis is typically characterized by headachewith an acute confusional state, with or without seizures. TheCalifornia Encephalitis Program identified the clinical profilesand etiologies of encephalitis in a large cohort of immunocompe-tent patients from 1998 to 2005 (7). A confirmed or probableetiology was identified in only 15.8% of cases; among these, vi-ruses were the causative agent in 68.5% of cases. Herpes simplexvirus is the most common cause of encephalitis in Western coun-tries (6, 53, 54). Enteroviruses, varicella-zoster virus, Epstein-Barrvirus, measles virus, and arboviruses, such as Japanese encephali-tis virus, West Nile virus, and Murray Valley encephalitis virus,have also been implicated as causes of encephalitis (6, 7, 54, 55).Beckham and Tyler provided a comprehensive list of other impor-tant and emerging viral causes of encephalitis (52). L. monocyto-genes may cause meningoencephalitis, a syndrome where featuresof both meningitis and encephalitis are present.

Burkholderia pseudomallei, the causal agent of melioidosis, has arelatively uncommon association with CNS involvement, ac-counting for around 4% of cases in northern Australia (56, 57),with distinctive clinical features being brain stem encephalitis, of-ten with cranial nerve palsies, together with peripheral motorweakness, or occasionally just flaccid paraparesis alone (myelitis),and presenting with urinary retention (5658). Fevers are usuallya prominent feature, and headaches are common, but neckstiffness is usually mild or absent. The computed tomography(CT) scan is often normal, but dramatic changes are seen onmagnetic resonance imaging, most notably a diffusely increasedT2-weighted signal in the midbrain, brain stem, and spinal cord,with variable symmetry and enhancement with gadolinium. CSFshows high protein, normal or slightly decreased glucose, and,most notably, a pleocytosis with lymphocytes usually predomi-nant. Bulbar palsy and decreased respiratory drive often necessi-tate ventilation, which may be required for many weeks (56, 5962). In northern Australia, patients infected with the flavivirusMurray Valley encephalitis virus can present with clinical featuresindistinguishable from those seen with melioidosis encephalomy-elitis.

Neurologic melioidosis is occasionally seen outside Australia,although it appears to be less common in melioidosis patients inSoutheast Asia than in Australia. Rather than the distinctive brainstem encephalitis and/or myelitis seen in Australia, patients withneurological melioidosis in Southeast Asia usually present with

Bacterial Mechanisms of Brain Invasion

October 2014 Volume 27 Number 4 cmr.asm.org 693

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

macroscopic cortical brain abscesses (63), suggesting that in thesecases there has been bacteremic spread to the brain. Frank men-ingitis is rarely seen in melioidosis. A recent report described thefirst case of neurological melioidosis in the United States, in a58-year-old male who presented with low-grade fever, nausea,vomiting, headaches, diplopia, left-sided ptosis, and transient ep-isodes of marked somnolence, after recent travel to Cambodia(64). CSF demonstrated increased levels of glucose, protein, andlymphocytes; however, microbiological investigation consistentlyshowed no organisms by Gram stain and failed to detect any or-ganisms on culture medium. B. pseudomallei was diagnosed onlyfollowing the culture of biopsy material collected during a suboc-cipital craniectomy. The mortality of neurological melioidosis isup to 30%, with ongoing residual neurological deficits in up to50% of patients overall (5658).

Focal Infections

The range of focal CNS infections includes brain abscesses, sub-dural empyema, and epidural abscesses. Brain abscess refers to anintracerebral infection that usually begins as localized cerebritis,which develops into a collection of pus that is surrounded by awell-vascularized capsule (65). The clinical presentations of brainabscesses are variable and depend on the anatomical area of thebrain that is compressed or affected. Brain abscesses commonlyoriginate from a contiguous focus of infection, such as the middleear, mastoid cells, or paranasal sinuses. Brain abscesses may alsooccur following (i) hematogenous spread of bacteria from a dis-tant focus of infection or (ii) brain trauma or neurosurgery (66). Arecent systematic review and meta-analysis of 123 brain abscessstudies reported between 1935 and 2012 demonstrated that Strep-tococcus and Staphylococcus spp. were the most frequently isolatedmicroorganisms, causing 34.9% and 18.9% of infections, respec-tively (67). Gram-negative enteric bacteria were responsible for15.0% of cases of brain abscess and were frequently associatedwith polymicrobial infections. Other, less common causativeagents include anaerobic bacteria (Bacteroides spp., Peptostrepto-coccus spp., and Fusobacterium spp.), Actinomycetales, Haemophi-lus spp., and Pseudomonas spp. (6668).

Subdural empyema refers to a collection of pus in the subduralspace, which is located between the dura mater and arachnoidmater layers of the meninges (see Barriers of the CNS, below). Incontrast, epidural abscesses are defined as a collection of pus be-tween the dura mater and the skull or vertebral column (69). Theetiologies of subdural empyema and epidural abscesses are similarto those of brain abscesses; however, due to the anatomical loca-tion of these infections, bacteria do not need to penetrate theblood-brain barrier (BBB) to establish disease.

BARRIERS OF THE CNS

The CNS is physically protected from injury by bony structures,such as the vertebrae and the cranium, and is also enveloped by themeninges and surrounded by CSF. The meninges comprise threelayers: the dura mater, arachnoid mater, and pia mater. The arach-noid mater and pia mater are separated by the subarachnoidspace, which is filled with CSF that is produced by the choroidplexus in the ventricles of the brain (Fig. 1) (70). The CSF providesmechanical support, regulates ion composition, and maintainschemical stability. Once it has circulated through the ventricles ofthe brain, the CSF enters the subarachnoid space and drains intothe blood through arachnoid villi and granulations. CSF also

drains through the cribriform plate into the lymphatics within thenasal mucosa (7173).

The complex functions of the brain are dependent on homeo-static mechanisms that regulate the ionic composition of the in-terstitial fluid that surrounds neurons in the parenchyma (74).This homeostasis is maintained by two cellular barriers which sep-arate the CNS from the systemic circulation: the BBB and thebrain-CSF barrier (BCSFB) (Fig. 1). These barriers protectthe brain from abrupt changes in blood biochemistry while at thesame time allowing delivery of nutrients and removal of metabo-lites. Importantly, these barriers also protect the CNS from inva-sion by microbes that may be present within the blood.

The Blood-Brain Barrier

The BBB (Fig. 1) exists at the level of cerebral microvessels andrepresents the largest interface for blood-brain exchange (75). TheBBB is composed of endothelial cells that line cerebral microves-sels in association with pericytes, astrocytes, and the basementmembrane (collectively referred to as a neurovascular unit, in as-sociation with neurons, microglia, and peripheral immune cells).Pericytes are attached to the abluminal surface of the endotheliumand, together with the astrocytic end feet, form a contiguousmembrane barrier. A physical barrier is formed at the interendo-thelial cleft by complex junctions that regulate the flux of ions,polar molecules, and macromolecules from the systemic circula-tion (74, 75).

At the interendothelial cleft, two types of junctions comprisethe BBB: adherens junctions and tight junctions. These junctionsblock the transport of a wide range of molecules, restricting para-cellular permeability across the BBB (75). In addition, endothelialcells forming the BBB lack fenestrations in their plasma mem-branes and have a reduced number of pinocytotic vesicles com-pared to endothelial cells in other tissues, which restricts transcel-lular flux (76). Despite this, certain solutes and macromoleculesmay be transported across the BBB via specific and nonspecificmechanisms, such as passive diffusion and receptor and/or ad-sorption-mediated transcytosis, or via ATP-binding cassettetransporters and solute carrier transporters, which are expressedon cerebral microvessels (75, 7779). The transcellular and para-cellular routes of entry to the CNS are highly relevant for micro-bial pathogens.

The Blood-Cerebrospinal Fluid Barrier

The BCSFB (Fig. 1) is formed by tight junctions between (i) epi-thelial cells located at the choroid plexus, in the lateral, third, andfourth ventricles of the brain; and (ii) endothelial cells of the veinsand venules within the subarachnoid space. Tight junctions be-tween the epithelial cells of the arachnoid mater also contribute tothe BCSFB by forming a barrier between the subarachnoid spaceand the leaky fenestrated blood vessels of the dura mater. Of theseBCSFB locations, the choroid plexus has a large vascular surfacearea and represents the major interface between fenestrated bloodvessels and CSF. Similar to the BBB, the BCSFB functions to pro-tect the CNS and helps to maintain homeostasis; however, thechoroidal epithelial cells of the BCSFB also secrete CSF from thechoroid plexus into the brain ventricular system, and the tightjunctions between the epithelial cells have a lower electrical resis-tance than that of BBB endothelial cells (80). Thus, the choroidplexus BCSFB may be more vulnerable to microbial penetrationvia paracellular mechanisms. The BCSFB at the arachnoid mater

Dando et al.

694 cmr.asm.org Clinical Microbiology Reviews

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

has a smaller total surface area than those of other barriers of theCNS and is not thought to be an important route for the entry ofsolutes into the brain (74). However, the arachnoid BCSFB mayrepresent an important site of microbial entry.

Immunosurveillance of the CNS

The brain parenchyma and spinal cord are populated throughoutby resident immune cells, the microglia, which are highly special-ized tissue macrophages that are maintained through in situ self-renewal without reconstitution from the bone marrow (8183).

Microglia continuously survey the brain parenchyma by samplingtheir microenvironment with highly motile processes and protru-sions (84). Recent data have also revealed that cells expressingCD11c are localized within the juxtavascular parenchyma of thehealthy mouse brain and extend processes to the glia limitans(astrocytic foot processes at the parenchymal basement mem-brane, surrounding the brain under the pia mater) (85). In agree-ment with a previous study (86), this raises the possibility thatdendritic cells, which are characterized by high-level CD11c ex-pression, may reside within the brain parenchyma. However, a

FIG 1 Anatomical locations and organizations of the blood-brain and blood-cerebrospinal fluid barriers and the olfactory portal. The skull and the meninges,which consist of the dura mater, arachnoid mater, and pia mater, protect the brain. The arachnoid mater and pia mater are connected by strands of connectivetissue called arachnoid trabeculae, which course through the CSF-containing subarachnoid space. At the cellular level, the BBB and BCSFB maintain homeostasisof the brain. The BBB is formed by tight junctions between endothelial cells lining cerebral microvessels, in addition to pericytes and astrocytes. The BCSFB isformed by tight junctions between (i) epithelial cells at the choroid plexus, (ii) endothelial cells of the veins and venules within the subarachnoid space, and (iii)the epithelial cells of the arachnoid mater. The olfactory system bypasses the cellular barriers of the CNS and provides a direct portal from the nasal cavity to theolfactory bulb within the brain.

Bacterial Mechanisms of Brain Invasion

October 2014 Volume 27 Number 4 cmr.asm.org 695

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

range of cells of the myeloid lineage (including activated micro-glia) also express CD11c, albeit at lower levels. Thus, the CD11c-expressing cells described within the brain parenchyma may alsorepresent a subpopulation of microglia (85).

Other resident immune cells of the CNS include perivascularmacrophages in addition to small populations of blood-deriveddendritic cells and macrophages within the choroid plexus andmeninges. Dendritic cells constitute approximately 1% of im-mune cells within the choroid plexus and meninges (87, 88), butunlike the putative dendritic cells of the brain parenchyma, cho-roid plexus and meningeal dendritic cells are distinct from micro-glia and closely resemble classical dendritic cells that require FMS-like receptor tyrosine kinase 3 ligand for development (88). TheCSF also contains a trafficking population of mononuclear cells,comprising T cells (90%), B cells (5%), monocytes (5%), and den-dritic cells (1%) (89).

Normal CNS immune surveillance occurs when centralmemory T cells migrate from the blood across the choroidplexus epithelium, enter the CSF, and scan the subarachnoidspace. In the absence of recognizing ligands presented by localantigen-presenting cells, T cells exit the subarachnoid spaceand drain to the secondary lymphoid organs via the nasal mu-cosa (90, 91). In contrast, recognition of presented antigensresults in the formation of immunological synapses and is ac-companied by cellular signaling that results in the recruitmentof peripheral inflammatory cells to the CNS via paracellular ortranscellular diapedesis (92).

The production of cytokines, free radicals (such as reactive ox-ygen species), and matrix metalloproteinases by inflammatorycells within the CSF spaces can subsequently result in degradationof the glia limitans, BBB/BCSFB dysfunction, and cellular death(9395). In bacterial meningitis, neuronal injury also occurs dueto the direct effects of bacterial toxins and virulence factors (9698). A recent study of human bacterial meningitis cases demon-strated that the profile of pro- and anti-inflammatory cytokinesand matrix metalloproteinases within the CSF is dependent uponthe causative pathogen (99). The sequelae of bacterial meningitisand the associated neuroinflammation include vasculitis and ce-rebral venous thrombosis, which can lead to cerebral ischemia. Anincrease in CSF outflow resistance in combination with interstitialedema results in raised intracranial pressure, which causes hy-poxic brain damage and may lead to death (100).

MICROBIAL ENTRY TO THE CNS

Microbial pathogens may access the CNS (i) by penetrating theBBB or the BCSFB or (ii) via the olfactory and/or trigeminalnerves (101). Peripheral nerves other than the olfactory and tri-geminal cranial nerves have also been implicated as routes of entryfor some viruses, but these are not reviewed here. Penetrations ofthe BBB and BCSFB represent common routes of CNS invasion,resulting in encephalitis or brain abscesses and in meningitis, re-spectively. In contrast to a diverse group of neurotropic viruses forwhich mechanisms are described for invasion of the brain via theolfactory sensory nerves, bacterial infection via the olfactory epi-thelium and the olfactory nerve bundles has been described infre-quently. Even less well understood is the fact that, in addition tothe olfactory nerve, the trigeminal nerve may also present a poten-tial pathway to the brain.

In Vivo and In Vitro Models of CNS Invasion

Much of our understanding of the mechanisms of microbial CNSinvasion has been obtained from in vivo and in vitro studies. Miceand rats are the most common animals used to model CNS infec-tions. These animal models have been used to investigate the sitesof microbial entry (20, 102106) and the roles of virulence deter-minants in CNS invasion and pathogenesis (107111). Despiteclear differences in the size and complexity of mouse and humanbrains, comparative transcriptomic studies between mice and hu-mans have demonstrated that gene expression profiles of thehealthy brain and choroid epithelium are highly conserved be-tween the species (112114).

There are, however, several important differences between themouse and human CNS that are relevant to microbial invasionstudies. In mice, the leptomeninges (comprising the arachnoidmater and pia mater) are significantly thinner and less vascularthan the case in humans (115). Additionally, the human choroidplexus epithelium has more abundant fibrovascular stroma alongthe vascular channels and a relatively larger surface area than thoseof the mouse choroid epithelium. Mice also possess very largeolfactory bulbs compared to humans (115), which may result in ahigher incidence of CNS invasion via the olfactory route than thecase in humans. Furthermore, mice lack several anatomically de-fined subclasses of astrocytes that are present in humans (116).These combined anatomical differences may influence the initialsite of microbial attachment and subsequent CNS invasion.

In vitro cell culture systems have been used to model the BBBand to elucidate the cellular and molecular mechanisms of micro-bial CNS invasion. Primary human, rodent, and bovine brain mi-crovascular endothelial cells have been used in some studies (97,117119); however, the isolation of these cells is technically demand-ing, and in vitro culture dramatically alters the transcriptome, leadingto downregulation of genes involved in BBB function (120, 121).Human umbilical vein endothelial cells (HUVECs) (122124),Caco-2 cells, and Madin-Darby canine kidney cells (125) have beenused as surrogate models of the BBB, although the noncerebral originof these cells may limit their relevance. The availability of immortal-ized brain endothelial cell lines (for example, human brain microvas-cular endothelial cells [HBMECs], hCMEC/D3, TY10, and BB19) hasenabled the human BBB to be studied. When grown on Transwellmembranes, HBMECs (i) form tight junctions, as measured by tran-sendothelial electrical resistance; (ii) express the adherens junctionand tight junction proteins zonula occludens-1, junctional adhesionmolecule A, claudin-5, occludin, and vascular endothelial cadherin;and (iii) demonstrate negligible paracellular permeability (126129).

It must be noted that brain microvascular endothelial cellsgrown in static monocultures represent a surrogate model for thehuman BBB, as they lack other components of the neurovascularunit that are required for the development of true BBB properties.Studies using pericyte-deficient mice have shown that pericytes(Fig. 1) are required for BBB integrity. Compared to wild-typemice, pericyte-deficient mice demonstrated an accumulation ofwater and tracer dyes within the brain, structural abnormalities incerebral endothelial tight junctions, and increased expression ofgenes known to increase vascular permeability, endothelial vesicletrafficking, and immune cell recruitment (130132). Astrocytesalso play a key role in regulating the BBB (133136), and as such,a range of coculture systems have been developed to more closelymodel the BBB properties of the neurovascular unit (137139).

Dando et al.

696 cmr.asm.org Clinical Microbiology Reviews

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

Choroid plexus epithelial cell lines that display the characteristicproperties of the BCSFB have been described (140, 141); however,the majority of in vitro microbial invasion studies have been per-formed using monocultures of brain microvascular endothelialcells.

BACTERIAL INVASION THROUGH THE BLOOD-BRAIN ANDBLOOD-CEREBROSPINAL FLUID BARRIERS

Sites of Entry

Classical bacterial meningeal pathogens may potentially enter theCSF by penetrating the BBB of cerebral microvessels and enteringthe extracellular fluid of the brain, which is continuous with theCSF. However, a more likely route involves direct entry to the CSFvia penetration of the BCSFB at either the arachnoid or the cho-roid plexus (142). The highly vascularized choroid plexus has beenfavored as a major site of bacterial entry to the CSF due to lowerelectrical resistances between choroid epithelial cells. Autopsies ofneonates who died from bacterial meningitis demonstrated thatmeningitis was frequently (84.0%) associated with an accumula-tion of a purulent exudate within the ventricles (ventriculitis) andthe choroid plexus (plexitis) (143). Ventriculitis preceding men-ingitis would support the role of the choroid plexus as the initialsite of bacterial invasion; however, ventriculitis frequently occursas a complication of meningitis, and the site of bacterial CNSinvasion remains incompletely understood.

In experimentally infected rhesus macaques, the choroid plexuswas the site where the earliest histopathologic lesions were ob-served in H. influenzae meningitis (144). Following intranasal in-oculation, H. influenzae was isolated from the CSF within the lat-eral cerebral ventricle, and this was associated with intravascularinflammatory infiltration and mild choroid plexitis (145). In 5- to7-week-old pigs, S. suis was localized within the choroid plexusand caused disruption of the brush border of the choroid epithe-lium and loss of the apical cytoplasm (146). Interestingly, autopsyof a 2-month-old infant who died of fulminant meningococcemiaprior to the onset of meningitis revealed adherent bacteria withinthe capillaries of the choroid plexus and, to a much lesser extent,the capillaries within the meninges (147). This would appear tosupport the choroid plexus as the preferential site of N. meningi-tidis entry to the CSF; however, no bacteria were found within orbetween choroid epithelial cells. In contrast, a study of autopsymaterial from symptomatic patients with meningococcal diseaserevealed the presence of N. meningitidis within the choroid plexusepithelium, interstitium, and blood vessels (148).

Neonatal rat models of E. coli meningitis have been used toidentify the potential site of entry to the CSF. Parkkinen et al.demonstrated that E. coli binds to the luminal surfaces of choroidepithelial cells, vascular endothelial cells, and ependymal cells lin-ing the brain ventricles (102). Similar findings were reported byZelmer et al., who suggested that E. coli K1 enters the CSF via thechoroid plexus, circulates to the subarachnoid space, and thenadheres to the meninges (103). In contrast, Kim et al. reported thatin the brains of 5-day-old rats, E. coli was localized within theperivascular areas of the subarachnoid space, not within the cho-roid plexus (20). Recently, the spatiotemporal events of CNS in-vasion were investigated in a BALB/c mouse model of pneumo-coccal meningitis (104). S. pneumoniae preferentially adhered tovessels within the subarachnoid space at 1 h postinfection andformed clusters around the endothelium at this location at later

time points. Remarkably, pneumococci could be detected in thechoroid plexus only at 8 h postinfection, suggesting that this is notthe site of initial entry to the CSF.

Mechanisms of Entry

Bacteria may cross the BBB and BCSFB to access the CNS bytranscellular penetration or paracellular entry or via infected leu-kocytes from the peripheral circulation (the Trojan horse mech-anism) (Fig. 2). Transcellular penetration occurs following bacte-rial adhesion to endothelial or epithelial cells. Bacteria aresubsequently translocated across these barriers by pinocytosis orreceptor-mediated mechanisms. Alternatively, bacteria may in-vade the CNS paracellularly following disruption of the tight junc-tions between cells forming the BBB and/or BCSFB, resulting inincreased permeability. Microorganisms may exploit more thanone of these mechanisms to access the CNS.

Bacterial attachment to HBMECs is a prerequisite for transcel-lular and paracellular penetration. There has been significant re-search into the host-pathogen interactions that occur prior to bac-terial invasion of brain microvascular endothelial cells (Table 1),including the identification of host receptors that mediate theseinteractions. E. coli, H. influenzae, N. meningitidis, and S. pneu-moniae bind to HBMECs via the 37/67-kDa laminin receptor(149152). The 37-kDa laminin receptor is a precursor for themature, 67-kDa laminin receptor, which binds laminin-1 withinthe basement membrane on the abluminal surface of eukaryoticcells (153). Kim et al. (150) demonstrated that the 67-kDa lamininreceptor is expressed on the basolateral side of HBMECs (i.e., theabluminal surface); however, cytoxic necrotizing factor 1 (CNF-1)of E. coli triggered clustering of the 67-kDa laminin receptor on

FIG 2 Mechanisms of blood-cerebrospinal fluid penetration by bacterialpathogens. Bacteria (purple) may invade the CNS via the BCSFB by transcel-lular penetration involving either pinocytosis or receptor-mediated mecha-nisms (A); by paracellular entry following the disruption of junctions (com-prising tight junctions and adherens junctions) between choroidal epithelialcells, endothelial cells of veins/venules within the subarachnoid space, or thecells of the arachnoid membrane (B); and by the Trojan horse mechanism,where microbes may transmigrate with infected leukocytes (such as macro-phages, as shown) (C).

Bacterial Mechanisms of Brain Invasion

October 2014 Volume 27 Number 4 cmr.asm.org 697

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

TA

BLE

1K

now

nba

cter

iall

igan

dsan

dth

eir

hos

tre

cept

ors

for

adh

esio

nto

and

inva

sion

ofth

ebl

ood-

brai

nba

rrie

ra

Bac

teri

alpa

thog

enL

igan

dH

BM

EC

rece

ptor

Fun

ctio

nR

efer

ence

(s)

Esc

heri

chia

coli

(an

dot

her

Gra

m-

neg

ativ

een

teri

cs)

Fim

HC

D48

Adh

esio

nto

HB

ME

Cs;

Fim

Hal

sopr

omot

esin

vasi

onby

acti

vati

onof

Rh

oA,w

hic

hin

duce

sh

ost

cyto

skel

etal

rear

ran

gem

ents

154,

410

Nlp

1U

nkn

own

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s;N

lp1

acti

vate

sh

ost

cyto

solic

phos

phol

ipas

eA

2

174

Om

pAgp

96h

omol

ogu

eLo

ops

1,2,

and

3of

Om

pAfu

nct

ion

inad

hes

ion

toan

din

vasi

onof

HB

ME

Cs;

Om

pA-m

edia

ted

inva

sion

isdu

eto

Rac

1ac

tiva

tion

and

acti

vati

onof

hos

tcy

toso

licph

osph

olip

ase

A2

161,

162,

173,

178

CN

F-1

37/6

7-kD

ala

min

inre

cept

orIn

vasi

onof

HB

ME

Cs;

CN

F-1

acti

vate

sR

hoA

and

recr

uit

sfo

cala

dhes

ion

kin

ase

toth

esi

teof

bact

eria

len

try

149,

150,

171,

172

IbeA

Vim

enti

n,P

SFIn

vasi

onof

HB

ME

Cs;

acti

vate

sR

ac1

178,

411

413

Ibe1

045

-kD

aIb

e10

rece

ptor

Inva

sion

ofH

BM

EC

s41

4,41

5A

slA

Un

know

nIn

vasi

onof

HB

ME

Cs

416

Tra

JU

nkn

own

Inva

sion

ofH

BM

EC

s41

7Y

ijPU

nkn

own

Inva

sion

ofH

BM

EC

s41

8Fl

agel

laU

nkn

own

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s41

9

Hae

mop

hilu

sin

fluen

zae

Ph

osph

oryl

chol

ine

PA

Fre

cept

orP

oten

tial

lyin

volv

edin

inva

sion

ofH

BM

EC

s42

0O

mpP

237

/67-

kDa

lam

inin

rece

ptor

Adh

esio

nto

HB

ME

Cs

151,

152

List

eria

mon

ocyt

ogen

esIn

lAE

-cad

her

inIn

vasi

onof

pola

rize

dh

um

anch

oroi

dpl

exu

sep

ith

elia

lcel

ls23

2In

lBgC

1q-R

,met

rece

ptor

tyro

sin

eki

nas

eIn

vasi

onof

HB

ME

Cs

and

pola

rize

dh

um

anch

oroi

dpl

exu

sep

ith

elia

lcel

ls21

3,23

2,42

1,42

2V

ipgp

96In

vasi

onof

the

brai

nin

vivo

160

IspC

Un

know

nA

dhes

ion

toan

din

vasi

onof

shee

pch

oroi

dpl

exu

sep

ith

elia

lcel

lsbu

tn

otH

BM

EC

s23

3

Myc

obac

teri

umtu

berc

ulos

isR

v098

0c,R

v098

7,R

v098

9c,

Rv1

801

Un

know

nIn

vasi

onof

HB

ME

Cs

118

HB

HA

Hep

aran

sulf

ate

glyc

osam

inog

lyca

ns

Adh

esio

nto

and

inva

sion

ofB

BM

EC

s23

4R

v031

1,R

v080

5,R

v093

1c,

Rv0

986,

MT

3280

Un

know

nIn

vasi

onof

HB

ME

Cs

235

Pkn

DLa

min

inA

ttac

hm

ent

to,i

nva

sion

of,a

nd

surv

ival

wit

hin

HB

ME

Cs

158

Nei

sser

iam

enin

giti

dis

Typ

eIV

pili

Un

know

nA

dhes

ion

toH

BM

EC

s;ty

peIV

pilu

s-m

edia

ted

adh

esio

nle

ads

toth

efo

rmat

ion

ofco

rtic

alpl

aqu

esan

dh

ost

mem

bran

epr

otru

sion

s12

2,12

4

PilQ

and

Por

A37

/67-

kDa

lam

inin

rece

ptor

Adh

esio

nto

HB

ME

Cs

151,

152

Opc

5

1in

tegr

in(m

edia

ted

bybi

ndi

ng

toth

ebr

idgi

ng

mol

ecu

les

vitr

onec

tin

and

fibr

onec

tin

)

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s;ac

tiva

tes

JNK

1,JN

K2,

p38,

and

prot

ein

tyro

sin

eki

nas

esi

gnal

ing

155,

191,

192

Stap

hylo

cocc

usau

reus

Ypf

Pan

dL

TA

Un

know

nIn

vasi

onof

HB

ME

Cs

423

Stre

ptoc

occu

sag

alac

tiae

Lmb

Lam

inin

Inva

sion

ofH

BM

EC

s15

9Ia

gAan

dL

TA

Un

know

nIn

vasi

onof

HB

ME

Cs;

IagA

anch

ors

LTA

toth

est

rept

ococ

calc

ellw

alla

nd

isre

quir

edfo

rin

vasi

on42

4

FbsA

Fibr

inog

enA

dhes

ion

toH

BM

EC

s15

6Sf

bAIn

tegr

ins

(via

S.ag

alac

tiae

bin

din

gto

imm

obili

zed

fibr

onec

tin

)In

vasi

onof

HB

ME

Cs

199

Dando et al.

698 cmr.asm.org Clinical Microbiology Reviews

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

the apical side (i.e., the luminal surface), where it could potentiallyinteract with bacteria within the cerebral microvasculature. In anin vivo model, Orihuela et al. reported that fluorescent micro-spheres coated with laminin receptor-binding adhesins (such ascholine-binding protein A [CbpA] of S. pneumoniae) adhered tothe cerebral endothelium of mice via the 37/67-kDa laminin re-ceptor following intravascular injection, as demonstrated by cra-nial window imaging (151). Several bacterial meningeal patho-gens have been shown to bind host glycoproteins, such asfibrinogen, vitronectin, fibronectin, laminin, and collagen, whichmay act as bridging molecules between the bacterium andHBMECs (109, 154159). In addition, the heat shock proteingp96 and a gp96 homologue act as receptors for the surface-ex-pressed virulence protein (Vip) of L. monocytogenes (160) andouter membrane protein A (OmpA) of E. coli (161, 162), respec-tively.

Transcellular Penetration of Brain MicrovascularEndothelial Cells

Transcellular penetration of brain microvascular endothelial cells,mainly via receptor-mediated mechanisms (Fig. 2), has been dem-onstrated for several bacteria, including E. coli, N. meningitidis, S.pneumoniae, group B streptococcus, S. suis, L. monocytogenes, andM. tuberculosis.

E. coli. E. coli translocates from the upper compartment to thelower compartment in an HBMEC Transwell model without increas-ing cellular permeability, thus supporting a transcellular mechanismof penetration (163). Electron microscopy studies have demon-strated that E. coli invades HBMECs through a zipper-like, recep-tor-mediated endocytosis mechanism (164). Intracellular E. coliE44 bacteria were observed in membrane-bound vesicles withinHBMECs at 30 min postinfection; by 45 min, the bacteria hadtranslocated to the basolateral side (164). Both early (early endo-some antigen 1 and transferrin receptor) and late (Ras-relatedprotein 7 and lysosome-associated membrane protein 1) endo-somal markers were recruited to vacuoles containing E. coli K1.However, the lysosomal hydrolytic enzyme cathespin D did notaccumulate within the vacuoles, demonstrating that lysosomal fu-sion was prevented. This was found to occur in a capsule-depen-dent manner (165).

Several E. coli proteins contribute to the adhesion to and inva-sion of HBMECs (Table 1). Attachment of E. coli to HBMECsinduces host cytoskeletal rearrangements and actin condensationbelow adherent bacteria (164). This is associated with a CNF-1-mediated recruitment of focal adhesion kinase and the cytoskel-etal protein paxillin to the 67-kDa laminin receptor, which formsclusters that colocalize with adherent E. coli (150). Interactionsbetween E. coli and HBMECs result in tyrosine phosphorylation offocal adhesion kinase and paxillin. The activity of focal adhesionkinase and its autophosphorylation site, tyrosine 397, were shownto be essential for E. coli invasion of HBMECs (166). Furthermore,the Src kinase-dependent activation of phosphatidylinositol 3-ki-nase and its interaction with focal adhesion kinase were requiredfor E. coli K1 invasion and host cytoskeletal rearrangement (167,168). Sukumaran and Prasadarao demonstrated that the associa-tion of phosphatidylinositol 3-kinase with focal adhesion kinaseresulted in the downstream activation of protein kinase C(PKC) in an Omp-A-dependent manner (169). Activated pro-tein kinase C was recruited to the E. coli entry site, where itinteracted with its substrate (myristoylated alanine-rich C kinase

PilA

2

1in

tegr

in(m

edia

ted

byS.

agal

acti

aebi

ndi

ng

toim

mob

ilize

dco

llage

n)

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s;P

ilAbi

ndi

ng

stim

ula

tes

foca

ladh

esio

nki

nas

eph

osph

oryl

atio

nan

ddo

wn

stre

amph

osph

atid

ylin

osit

ol3-

kin

ase

acti

vati

on

109,

425

PilB

Un

know

nIn

vasi

onof

HM

BE

Cs

425

Srr-

1Fi

brin

ogen

(Srr

-1bi

nds

amin

oac

ids

283

to41

0of

the

fibr

inog

en

chai

nvi

aa

dock

,loc

k,an

dla

tch

mec

han

ism

)

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s20

0,20

1,42

6

Hvg

AU

nkn

own

Adh

esio

nto

HB

ME

Cs

198

AC

PG

lyco

sam

inog

lyca

ns

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s;in

vasi

onpo

ten

tial

lyoc

curs

follo

win

gR

ho

GT

Pas

e-de

pen

den

tac

tin

rear

ran

gem

ents

157,

427

Stre

ptoc

occu

spn

eum

onia

eC

bpA

37/6

7-kD

ala

min

inre

cept

orA

dhes

ion

toH

BM

EC

s15

1P

hos

phor

ylch

olin

eP

AF

rece

ptor

Inva

sion

ofH

BM

EC

s;bi

ndi

ng

topl

atel

etac

tiva

tin

gfa

ctor

rece

ptor

indu

ces

colo

caliz

atio

nof

-a

rres

tin

193,

197

Nan

AU

nkn

own

Adh

esio

nto

and

inva

sion

ofH

BM

EC

s19

4,19

5a

AC

P,a

lph

aC

prot

ein

;Asl

A,a

ryl-

sulf

ates

ulf

ohyd

rola

se;B

BM

EC

,bov

ine

brai

nm

icro

vasc

ula

ren

doth

elia

lcel

ls;C

bpA

,ch

olin

ebi

ndi

ng

prot

ein

A;C

NF-

1,cy

toto

xic

nec

roti

zin

gfa

ctor

1;Fb

sA,fi

brin

ogen

-bin

din

gpr

otei

n;F

imH

,typ

e1

fim

bria

ladh

esin

;HB

HA

,hep

arin

-bin

din

gh

emag

glu

tin

inad

hes

in;H

BM

EC

s,h

um

anbr

ain

mic

rova

scu

lar

endo

thel

ialc

ells

;Hvg

A,h

yper

viru

len

tgr

oup

Bst

rept

ococ

cus

adh

esio

n;I

beA

,in

vasi

onof

brai

nen

doth

elia

lcel

lpro

tein

A;I

agA

,in

vasi

on-a

ssoc

iate

dge

ne

A;I

nlA

,in

tern

alin

A;I

nlB

,in

tern

alin

B;L

TA

,lip

otei

choi

cac

id;L

mb,

lam

inin

-bin

din

gpr

otei

n;N

anA

,neu

ram

inid

ase

A;N

lp1,

new

lipop

rote

in1;

Om

pP2,

oute

rm

embr

ane

prot

ein

P2;

Opc

,ou

ter

mem

bran

epr

otei

nC

;PA

F,pl

atel

etac

tiva

tin

gfa

ctor

;PilQ

,pilu

sse

cret

inpr

otei

n;P

orA

,maj

orou

ter

mem

bran

epr

otei

n;P

SF,p

olyp

yrim

idin

e-tr

act-

bin

din

gpr

otei

n(P

TB

)-as

soci

ated

splic

ing

fact

or;S

fbA

,str

epto

cocc

alfi

brin

ogen

bin

din

gpr

otei

nA

;Srr

-1,s

erin

e-ri

chre

peat

1;V

ip,v

iru

len

cepr

otei

n.

Bacterial Mechanisms of Brain Invasion

October 2014 Volume 27 Number 4 cmr.asm.org 699

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

substrate), leading to an accumulation of actin (169). The activatedprotein kinase C also interacted with caveolin-1 colocalized withcondensed actin underneath the bacterial entry site to form caveolae,which are plasma membrane invaginations that are involved in en-docytosis and signal transduction (170). CNF-1 (via the 37/67-kDalaminin receptor) and type 1 fimbrial adhesin (FimH; via CD48) havealso been shown to induce cytoskeletal rearrangements through acti-vation of the GTPase RhoA (149, 171, 172).

In an independent mechanism, E. coli OmpA and new lipopro-tein 1 (Nlp1) promote the activation of host cytosolic phospho-lipase A2 (173, 174). The activation of cytosolic phospholipase A2generated arachidonic acid metabolites, induced host actin cyto-skeletal rearrangements, and was essential for E. coli K1 invasionof HBMECs (173, 175177). OmpA and invasion of brain endo-thelial cell protein A (IbeA) also trigger the phosphorylation ofSTAT3, which results in the activation of Rac1, a Rho familyGTPase that regulates host cytoskeletal rearrangements (178).Notably, OmpA is highly conserved between Gram-negative bac-teria and may play a role in CNS invasion by those whose mecha-nisms of entry are unknown. Similarly, a B. pseudomallei toxin,Burkholderia lethal factor 1 (BLF-1), is structurally similar to E.coli CNF-1 and possesses a number of conserved residues that, inCNF-1, promote deamidation of glutamine and result in the acti-vation of RhoA and subsequent host cytoskeletal rearrangements(179). Thus, OmpA and BLF-1 may be attractive targets for futurestudies of the mechanisms of B. pseudomallei CNS invasion.

N. meningitidis. The attachment of N. meningitidis toHBMECs is mediated by type IV pili. This initial attachment isinhibited by high cerebral microcirculation shear stress levels;however, once attached, N. meningitidis proliferates and is able toresist high blood velocities by forming microcolonies with strongbacterium-bacterium interactions (124) and bacterium-host interac-tions (122). CD46 was previously identified as the receptor for type IVpili (180); however, this was not corroborated by additional studies,and it is likely that the type IV pilus attaches to host cells indepen-dently of CD46 (181). The type IV pilus protein PilQ, in addition tothe outer membrane porin PorA, was demonstrated more recently tobind to the 37/67-kDa laminin receptor on HBMECs (151). Further-more, studies with human bronchial epithelial cells show that themeningococcal type IV pilus binds to the platelet activating factor(PAF) receptor and that synergy between a pilin-linked glycan andphosphorylcholine decorating moieties is required for pili to effi-ciently engage the receptor (182).

N. meningitidis is thought to invade HBMECs primarily byparacellular mechanisms (see below); however, in vitro studieshave demonstrated that small numbers of both encapsulated andnonencapsulated meningococci are internalized by HUVECs andHBMECs (183185). Following initial bacterial attachment, themeningococcal minor pilin protein, PilV, triggers a cellular re-sponse in which endothelial cells form protrusions around N.meningitidis (122). These cellular projections further protect thebacterial microcolony from the mechanical stresses associatedwith high-velocity blood flow (122) and also lead to the engulf-ment and internalization of N. meningitidis (186). The host sig-naling events that contribute to these interactions have beenelucidated. Following type IV pilus-mediated attachment, N.meningitidis activates the host cell 2-adrenoceptor, leading to thetranslocation of -arrestin to the site of bacterial attachment at theplasma membrane (187). Several host proteins, including ezrinand moesin, which regulate the cortical cytoskeleton through F-

actin-binding sites, are then recruited to the site of bacterial at-tachment and accumulate in honeycombed molecular complexes,referred to as cortical plaques, underneath the meningococcal mi-crocolonies (186, 188, 189). The translocation of -arrestin alsoresults in the docking and activation of the protein tyrosine kinaseSrc, which phosphorylates cortactin (187, 188). Cortical actin isthen polymerized in a Rho GTPase- and Cdc42-dependent man-ner, which leads to the formation of cell membrane protrusions(186). In human bone marrow endothelial cells, the recruitmentof cortactin and formation of membrane protrusions were alsoshown to be dependent on the activation of a phosphoinositide-3-kinase/Rac1 signaling pathway by lipooligosaccharide (190).

Once internalized, the membrane-bound vesicles containingmeningococci associate with transferrin receptor and lysosome-associated membrane protein 1 endosomal markers (185). It islikely that lysosomal fusion is subverted, as live meningococcihave been shown to translocate from the basolateral side to theapical side in a Transwell BCSFB invasion model (140). In vitrostudies have demonstrated that capsular polysaccharide inhibitsthe invasion of N. meningitidis into HBMECs (191) and humanchoroid plexus epithelial cells (140) but is essential for survivaland replication within the intracellular niche (185).

For nonencapsulated meningococcal strains, the invasion ofHBMECs is dependent upon outer membrane protein C (Opc).Opc binds to the human serum factor fibronectin, which acts as abridging molecule and anchors the bacterium to the 51 integrinreceptor of HBMECs (191). This interaction results in the activa-tion of mitogen-activated protein kinase (c-Jun N-terminal kinase1 [JNK1], JNK2, and p38) and protein tyrosine kinase signal trans-duction pathways (192). Pretreatment of cells with specific JNK1,JNK2, and protein tyrosine kinase inhibitors significantly reducedthe internalization of N. meningitidis by HBMECs, without affect-ing bacterial adherence. Blocking of the 51 integrin receptor ofHBMECs also decreased JNK activation in the presence of N. men-ingitidis. Furthermore, the use of Opc-deficient mutant strainsdemonstrated that JNK signaling, but not p38 signaling, was me-diated by Opc expression (192). A more recent study identifiedthat Opc preferentially binds to activated vitronectin within hu-man serum and that this interaction promotes N. meningitidisinvasion of HBMECs (155). In vitro, the enhanced invasion ofnonencapsulated N. meningitidis strains is thought to be due to theunmasking of Opc; however, the role of Opc in vivo has yet to bedetermined.

S. pneumoniae. S. pneumoniae is internalized by human and ratbrain microvascular endothelial cells via receptor-mediated endo-cytosis. This uptake does not involve the formation of cellularmembrane protrusions as discussed for N. meningitidis (193). Theadhesion of pneumococci to HBMECs is mediated by interactionsbetween CbpA and the 37/67-kDa laminin receptor (151) andbetween neuraminidase A (NanA) and an unknown cellular re-ceptor(s) (194). Uchiyama et al. demonstrated that NanA, a sur-face-anchored sialidase, was necessary and sufficient to promotepneumococcal adhesion to and invasion of HBMECs (195). Fol-lowing initial attachment, the laminin G-like domain of NanAinitiates chemokine signaling and inflammatory activation of en-dothelial cells (194). The activation of endothelial cells results inthe increased expression of PAF receptorthe receptor for pneu-mococcal phosphorylcholine, which is a component of the cellwall and acts as a molecular mimic of the chemokine PAF (196).

In HBMECs, S. pneumoniae within vacuoles may (i) transit

Dando et al.

700 cmr.asm.org Clinical Microbiology Reviews

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

through the cell to the basolateral surface, (ii) recycle back to theapical surface, or (iii) be killed within the vacuole (193). Opaque S.pneumoniae variants, which express more capsular polysaccharideand less phosphorylcholine than transparent variants, were effi-ciently killed, suggesting that vacuoles containing these strainswere targeted for lysosomal fusion. In contrast, transparent pneu-mococci may undergo some recycling back to the apical surface;however, by 5 h postinfection in a Transwell system, the majorityof bacteria had translocated to the basolateral chamber (193). Theinvasion of HBMECs by transparent S. pneumoniae depends onphosphorylcholine, the PAF receptor, and -arrestin, which tar-gets G-protein-coupled receptors (such as PAF receptor) to clath-rin-coated vesicles (193, 197). S. pneumoniae induces the translo-cation of -arrestin from the cytosol to the plasma membrane,where it colocalizes with the PAF receptor. This interaction alsostimulates mitogen-activated protein kinase signaling, which isrequired for pneumococcal uptake (197). Vacuoles containing S.pneumoniae colocalized with early and late endosomal markers;however, increased expression of -arrestin subverted these vac-uoles from lysosomal trafficking and promoted the transcytosis ofviable bacteria. The small number of pneumococcus-containingvacuoles that underwent recycling to the apical surface demon-strated colocalization with Ras-related protein 11, which regulatesendosome recycling. These findings suggest that the pneumococ-cus-induced interactions between PAF receptor and -arrestincontribute to the transcellular penetration of HBMECs by S. pneu-moniae (197).

Group B streptococcus. Several group B streptococcal proteinspromote adhesion to and invasion of HBMECs (Table 1). The hyper-virulent group B streptococcus adhesin (HvgA) was identified as asequence type 17 (ST-17)-specific virulence factor which is anchoredto the group B streptococcal cell wall by sortase A (198). ST-17 is aclonal complex belonging to the group B streptococcus capsular se-rotype III and is responsible for 80% of neonatal meningitis cases.Therefore, it was proposed that HvgA expression may contribute tothe hypervirulence of ST-17. In vitro studies using an isogenic hvgAmutant demonstrated that HvgA was required for efficient group Bstreptococcal adhesion to HUVECs, primary rodent choroid plexusepithelial and brain microvascular endothelial cells, and immortal-ized HBMECs. Furthermore, in a murine model of hematogenousmeningitis, HvgA was required for CNS invasion, although it had noeffect on the levels of bacteremia (198).

A common characteristic of several other group B streptococ-cal adhesins/invasins is the ability to bind components of theHBMEC extracellular matrix, such as laminin, collagen, and fi-bronectin (109, 157, 159, 199). The group B streptococcal pilus tipadhesin (PilA) and the recently identified streptococcal fibronec-tin-binding protein A (SfbA) bind immobilized collagen and fi-bronectin, respectively. The collagen and fibronectin then associ-ate with HBMECs via integrins, and these interactions facilitatethe entry of group B streptococcus (109, 199). Similarly, the groupB streptococcal serine-rich repeat 1 (Srr-1) glycoprotein binds thefibrinogen A chain within human blood through a dock, lock,and latch mechanism (200, 201). During this interaction, fi-bronectin docks between two IgG-like folds (N2 and N3 domains)of the binding region of the adhesin, which initiates a conforma-tional change whereby a flexible region of the N3 domain latchesto form a -strand and completes a -sheet within the N2 domain,effectively locking the ligand in place (200). Seo et al. demon-strated that deletion of the latch-like domain of the C-terminal

end of the Srr-1 fibrinogen-binding region significantly reducedgroup B streptococcal adhesion to HBMECs (201). The fibrino-gen-binding protein (FbsA) also mediates group B streptococcaladherence to HBMECs, by binding immobilized fibrinogen (156),although it is unknown if this interaction also occurs via a dock,latch, and lock mechanism.

Electron microscopy studies have demonstrated that in vitro,group B streptococcus associates closely with the cell membraneof HBMECs and is enveloped by microvillous structures (202).Group B streptococci are internalized within membrane-boundvesicles and translocate from the apical side to the basolateral sideof HBMECs without a marked change in transendothelial electri-cal resistance. Serotype III group B streptococci were shown toinvade HBMECs more efficiently than representative strains ofserotypes Ia, Ib, II, and V; however, the serotype III capsule itselfdid not facilitate group B streptococcal invasion (202). Internal-ized group B streptococci did not undergo significant replicationand survived within HBMECs for up to 20 h (202). Intracellularsurvival of group B streptococcus may be promoted by the CiaR-CiaH two-component regulatory system, which regulates severalgenes associated with stress tolerance and the subversion of hostdefenses (203). A serotype III strain that was deficient in the CiaRresponse regulator demonstrated adhesion and invasion levelssimilar to those of the wild-type group B streptococcus inHBMECs but was associated with a significant decrease in intra-cellular survival. Broth inhibition assays demonstrated that CiaRconferred resistance to antimicrobial peptides, lysozyme, and re-active oxygen species (203).

The host signal transduction pathways that are involved in theuptake of group B streptococcus by HBMECs are similar to thosediscussed for E. coli. In vitro, the trimolecular interactions betweenPilA, collagen, and 21 integrins on HBMECs stimulate the phos-phorylation of focal adhesion kinase at tyrosine 397, which then in-duces host cytoskeletal rearrangements and the uptake of group Bstreptococci via the recruitment and activation of phosphatidylinosi-tol 3-kinase and paxillin (109, 204). It has also been demonstratedthat group B streptococcus invasion occurs via cytoskeletal rear-rangements that are induced following the activation of RhoA andRac1 (205). Furthermore, group B streptococcus induces serine 505phosphorylation of host cytosolic phospholipase A2, which leads tothe release of arachidonic acid metabolites, including cysteinyl leuko-trienes (206). In vitro pharmacological inhibition studies demon-strated that the activation of cytosolic phospholipase A2 was requiredfor efficient group B streptococcal invasion of HBMECs. In addition,brain colonization by group B streptococcus was significantly re-duced in mice deficient in cytosolic phospholipase A2 compared towild-type animals in a model of hematogenous meningitis. Followingcytosolic phospholipase A2 activation and the release of cysteinyl leu-kotrienes, downstream signaling activates protein kinase C, which isinvolved in the regulation of actin cytoskeletal rearrangements (206).

S. suis. The zoonotic pathogen S. suis adheres to but does notinvade HBMECs in vitro (207). In contrast, invasion of porcinebrain microvascular endothelial cells has been observed for thisswine pathogen. Inhibition studies demonstrated that S. suis inva-sion of porcine brain microvascular endothelial cells required ac-tin filaments but not microtubular cytoskeletal elements or activebacterial RNA or protein synthesis (208). Electron microscopy stud-ies showed that S. suis adhered to porcine brain microvascular en-dothelial cells at 5 min postinfection, and at 2 h postinfection, thestreptococci were in close contact with the cells, within invagi-

Bacterial Mechanisms of Brain Invasion

October 2014 Volume 27 Number 4 cmr.asm.org 701

on June 30, 2019 by guesthttp://cm

r.asm.org/

Dow

nloaded from

http://cmr.asm.orghttp://cmr.asm.org/

nated cell membrane structures and underneath the cell surface,behind the cell membrane. Unlike the case of group B strepto-cocci, following internalization within membrane-bound vacu-oles, the number of viable intracellular S. suis organisms steadilydecreased over time, and viable cocci were not detected after 7 h(208). However, similar to the case with S. pneumoniae and groupB streptococcus, the capsular polysaccharide of S. suis partiallyinhibited interactions with brain microvascular endothelial cells(208).

The S. suis proteins and virulence factors that mediate invasionof porcine brain microvascular endothelial cells remain largelyunknown. Pretreatment of porcine brain microvascular endothe-lial cells with S. suis lipoteichoic acid reduced but did not abolishthe adhesion and invasion of S. suis, in a dose-dependent manner,suggesting that lipoteichoic acid may play a role in S. suis interac-tions with porcine brain microvascular endothelial cells (209).Vanier et al. screened a transposon mutagenesis library of S. suisand identified several genes that may contribute to the invasion ofporcine brain microvascular endothelial cells (210). These geneswere characterized as encoding proteins belonging to the follow-ing groups: surface proteins, transport/binding proteins, regula-tory functions, metabolism, amino acid synthesis, protein synthe-sis, and hypothetical proteins. However, the roles of these genes inS. suis invasion of porcine brain microvascular endothelial cellshave not been confirmed using isogenic mutants.

As discussed above, the choroid plexus may be the site of entryto the CNS for S. suis. In order to study the interactions between S.suis and choroid plexus epithelial cells, Tenenbaum et al. devel-oped an inverted Transwell model in which the translocation of S.suis from the basolateral, blood side to the apical, CSF sidecould be investigated (211). Following S. suis infection of the ba-solateral side, the transepithelial electrical resistance and paracel-lular permeability remained constant for up to 4 h. S. suis invadedporcine choroid plexus epithelial cells from the basolateral side inthe inverted Transwell system; however, invasion was rare when S.suis was inoculated into the apical chamber in the standard Transwellmodel. These findings were also replicated in a human choroid plexusepithelial cell inverted Transwell model (140). This suggested that S.suis may interact with components of the extracellular matrix thatare accessible on the basolateral side. The translocation of S. suisthrough porcine brain microvascular endothelial cells was inhib-ited when cells were treated with a phosphatidylinositol 3-kinaseinhibitor, demonstrating that phosphatidylinositol 3-kinase maybe involved in the uptake of S. suis (211).

L. monocytogenes and B. pseudomallei. L. monocytogenes canenter the CNS by the Trojan horse mechanism (see below) or bytranscellular penetration of HBMECs. Using HUVECs andHBMECs, it was demonstrated that L. monocytogenes can invadeendothelial cells directly or via cell-to-cell spread from adherent,infected mononuclear phagocytes (212, 213). In HUVECs, L.monocytogenes attaches to the cellular surface and induces mem-brane ruffling, which leads to internalization (214). In HBMECs,L. monocytogenes invasion is preceded by intimate interactionsbetween the bacterium and microvilli on the cell surface (215).The invasion of L. monocytogenes into HBMECs requires actinmicrofilaments (213), but unlike the case for other meningealpathogens, it does not involve the activation of phosphatidylino-sitol 3-kinase (213) or cytosolic phospholipase A2 (175).

Once internalized, L. monocytogenes degrades the phagosomevia the activity of a pore-forming toxin (listeriolysin) and two

phospholipases (PlcA and PlcB), and it escapes into the cytoplasmby using actin-based motility, which is driven by the actin assem-bly-inducing protein, ActA. Following escape into the cytoplasm,L. monocytogenes can replicate within HUVECs and HBMECs anduse actin-based motility to spread to adjacent cells (212, 213), thusavoiding an extracellular lifestyle. However, L. monocytogenes canbe isolated from the CSF (216), suggesting that translocationthrough the cellular barriers of the CNS also occurs.

B. pseudomallei has an intracellular lifestyle similar to that of L.monocytogenes: the type III secretion system of B. pseudomallei(particularly the BopE and BopA effector proteins and the Bsatranslocation apparatus) is required for escape from the vacuoleand the subversion of autophagy (217222). In an epithelial cellline or a murine macrophage-like cell line, cell-to-cell spread thenoccurs by cell fusion, with the formation of multinucleated giantcells, in a type VI secretion-dependent process. BimA, which isnecessary for actin-mediated motility, and Fla2, which is thoughtto mediate intracellular motility, are also required and are consid-ered to mediate cell-to-cell contact prior to cell fusion (223227).To our knowledge, the ability of B. pseudomallei to directly invadeHBMECs has not been investigated, but it would be reasonable tohypothesize that the intracellular lifestyle of B. pseudomallei maycontribute to the pathogenesis of CNS invasion. Interestingly, arecent study investigated the variable virulence factors of B. pseu-domallei associated with melioidosis in Australia and reportedthat B. pseudomallei strains harboring a bimA allele that shares95% homology with B. mallei bimA (bimABm) (228) were signifi-cantly associated with neurological melioidosis (229). Patientsthat were infected with B. pseudomallei harboring the bimABmallele were found to be 14 times more likely to present with neu-rological involvement than patients infected with strains harbor-ing the B. pseudomallei bimA variant (bimABp) (229), indicating arole for actin-mediated motility in