University of Groningen

The type IV secretion systems of Neisseria gonorrhoeaePachulec, Emilia

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l

Summary and concluding remarks

Secreted substrates of Gram negative bacteria have to pass both the inner and the outer

membrane. To accomplish this, these bacteria have developed different secretion

mechanisms. To date at least six different types of secretion systems have been identified

which are briefly discussed in chapter 1 with an emphasis on their function, type of secreted

molecules and their role in pathogenesis. Among these systems, the type IV secretion systems

(T4SSs) are the most versatile and divergent in term of both prevalence and function. They

are present in Gram negative and positive bacteria, in Archaea, and in wall-less bacteria [I].

T4SSs are ancestrally related to the plasmid conjugation systems, and still the majority of

known T4SSs is plasmid encoded and responsible for conjugative transfer of DNA. Transfer

of DNA via conjugation is one of the major causes of rapid horizontal gene transfer and leads

to the rapid spread of antibiotic resistance. A prototype of conjugative T4SSs is encoded by

the F plasmid of E. coli and has been described in great detail [2]. In A. tumefaciens DNA

and effector molecules are transported via a T4SS resulting in crown gall tumors. The vir

system of A. tumefaciens is the prototype of an effector trans locator T4SSs, and has also been

studied in great detail [3-4]. Over the last decades, increasing numbers ofT4SS systems have

been identified. Many of these systems have a sim ilar organization and architecture as the vir

system of A. tumefaciens. Several pathogenic bacteria utilize T4SSs to secrete virulent

macromolecules that alter the host cellular response. Examples of these are the cag system of

H. pylori, the ptl system of B. pertussis and the dot/icm of L. pneumophila that allow the

bacteria to survive inside their host cells. Other T4SSs are adapted to divergent functions

including DNA uptake from the medium (comB system of H. pylori) or the release of DNA

into the medium (T4SS of N gonorrhoeae). During the work described in this thesis the

T4SSs of Neisseria gonorrhoeae were studied. N. gonorrhoeae is a Gram negative aerobic

diploccocus. It is a human pathogen causing gonorrhea, the second most common sexually

transmitted disease in the world. Although antibiotic therapy is still effective, the rapid rise of

antibiotic resistance has resulted in strains that are increasingly resistant to the available

treatments. The rapid spread of antibiotic resistance in gonoccoci is a consequence of highly

efficient horizontal gene transfer resulting in a panmicitic gonoccocal population structure. N.

gonorrhoeae encodes two types of T4SSs. A conjugative T4SS located on a gonococca'i

conjugative plasmid and a T4SS involved in DNA release encoded within the gonococcal

genetic island (GGI). The functional and organizational characteristic of both systems are the

subject ofthis thesis.

In chapter 2, the T4SS encodt

GGI is a 57 kb chromosom,

proximity of the chromosome

unique T4SS, which secretes

for transformation of other got

fold [5]. In order to idel1ltify al

mutational studies were perfc

parA [5] and parB genes were

region, could be deleted withe

by the first operon (traD, In

studies of the second operon ~

ycb and ych were crucial for [

traA gene encodes the TraA I

showed that the MS II A strai

results in a truncated form 0

contain a full length version,

tested. Surprisingly, the trune

secretion was inhibited by th

peptidase-like serine protease

also led to defective pilin as~

transport in T4SSs can occu

length version of TraA, CO

encoded by the GGI with

chromosomal markers, but n(

found in a small subset of ~

contain the traA mutation d

conjugation.

The conjugative T4SS of

gonoccocal conjugative pla~

plasmid with no detectable

determinant [9]. Restriction

plasm ids from different isola

plasm ids [10] , which were n

142

the inner and the outer

led different secretion

ms have been identified

unction, type of secreted

ipe IV secretion systems

~nce and function. They

In wall-less bacteria [I].

md still the majority of

msfer of DNA. Transfer

I gene transfer and leads

Ie T4SSs is encoded by

in A. tumefaciens DNA

/IT gall tumors. The vir

4SSs, and has also been

; ofT4SS systems have

d architecture as the vir

:Ss to secrete virulent

e are the cag system of

'1lophila that allow the

to divergent functions

or the release of DNA

ibed in this thesis the

}ram negative aerobic

lost common sexually

~ctive, the rapid rise of

stant to the available

:onsequence of highly

)pulation structure. N

lted on a gonococcal

'ithin the gonococcal

, both systems are the

Chapter 5

In chapter 2, the T4SS encoded within the gonoccocal genetic island (GG!) was studied. The

GGI is a 57 kb chromosomally encoded genetic island inserted at the dif site in close

proximity of the chromosomal origin of replication (oriC). A part of the GGI encodes a

unique T4SS, which secretes DNA into the extracellular milieu. The secreted DNA is used

for transformation of other gonococci, increasing the rate of DNA transfer approximately 500

fold [5]. In order to identifY all the genes within the GGI, that are involved in DNA secretion,

mutational studies were performed. Studies on the expl-parA region showed that only the

parA [5] and parB genes were essential for DNA secretion, whereas all the other genes in this

region, could be deleted without any effect. Previous reports showed that two genes encoded

by the first operon (traD, traJ) are essential for DNA secretion [6-7]. Further mutational

studies of the second operon showed that all genes with an exception for traA, ybe, trbI, ybi,

ycb and ych were crucial for DNA secretion by gonococci [5 , 8, this thesis]. Interestingly, the

traA gene encodes the TraA pilin which is not essential for DNA secretion . Further analysis

showed that the MS II A strain contains a frame shift mutation within the traA gene which

results in a truncated form of the protein. Remarkably, the majority of gonoccocal strains

contain a full length version of TraA. The effect of both traA fomls on DNA secretion was

tested . Surprisingly, the truncated version of traA does not affect DNA secretion while DNA

secretion was inhibited by the presence of a full length protein . Mutations in TrbI , a leader

peptidase-like serine protease, which catalyzes the circularization of the cyclic pilin subunit

also led to defective pilin assembly and to DNA secretion. This demonstrated that substrate

transport in T4SSs can occur in the absence of pilus. To examine the function of the full

length version of TraA., conjugation assays were performed to test whether the T4SSs

encoded by the GG! with a full length version of TraA mediate conjugal transfer of

chromosomal markers, but no conjugation could be detected. Thus secretion of DNA is only

found in a small subset of strains, which contain a mutation in TraA. Strains that do not

contain the traA mutation do not secrete DNA, but also do not seem to be involved in

conjugation.

The conjugative T4SS of N gonorrhoeae was studied in chapter 3. Three types of

gonoccocal conjugative plasm ids have been described in N gonorrhoeae; a 24.5 MDa

plasmid with no detectable marker, and two 25 .2 MDa plasm ids which contain the tetM

determinant [9]. Restriction endonuclease mapping and Southern blotting of conjugative

plasm ids from different isolates revealed two different 25.2 MDa tetM containing conjugative

plasmids [10], which were named the "American" and " Dutch" type plasmid [111. In chapter

"J 4 .\

!

Summary and concluding remarks

3 tetracycline conjugative plasmids with a Dutch type backbone were identified in a large

percentage of clinical isolates. The complete sequence of the Dutch type gonoccoccal

tetracycline plasmid was determined. Within plasm ids with a Dutch type backbone insertions

of both Dutch and American tetM determinants were found . Next to the insertion of the

different tetM determinants, the only difference between the strains with the tetM determinant

(25.2 MDa plasm ids) and strains without the tetM determinant (24.5 MDa plasm ids) was the

insertion of the ngoSKl1375 gene. The conjugative plasmids with the Dutch type backbone

contained similarly to other IncPI plasm ids backbone modules for replication initiation,

conjugative DNA transfer, mating-pair formation, stable plasmid inheritance and control.

Genes encoded in the backbone modules of the Dutch type neisserial conjugative plasmids

are phylogenetically divergent from the other IncP I subfamilies, suggesting that they are

members of a novel IncPI subfamily. Interestingly, the Dutch type neisserial conjugative

plasm ids contain a 'genetic load', module not found in other IncPl plasmids. Within this

region three different putative toxin/antitoxin systems are located. Remarkably, two of the

toxin/antitoxin systems belong to the ZetalEpsilon toxin/antitoxin family. Therefore, Dutch

type neisserial conjugative plasm ids are a unique example of multiple copies of

toxin/antitoxin systems. A third toxin/antitoxin system identified on the neisserial conjugative

plasmid belongs to the VapD family. The vapD gene has been associated with virulence in

several pathogenic bacteria [12-14J but its precise role is still unknown. In H injluenzaea the

vapX gene was identified in the same operon as vapD and was shown to function as an

antitoxin for VapD [15]. A homologue of the VapX protein was also found next to the vapD

gene on the cryptic pJDl plasmid of N. gonorrhoeae [16J. Remarkably, the Dutch type

conjugative plasmid of N. gonorrhoeae contained only the vapD gene. Interestingly, the

expression of the VapX antitoxin protein of pJD 1 in E. coli acceptor cells strongly increased

the transfer of the conjugative plasmid from N. gonorrhoeae to E. coli, indicating that the

VapX protein located on pJDl might function as an antitoxin for VapD on the conjugative

plasmid. This might explain the limited host range of neisserial conjugative plasmids. Most

plasmids contain only a single toxin/antitoxin system, and the advantage of having three

different toxin/antitoxin systems on one conjugative plasmid remains unclear. Comparison of

the T4SS system encoded by the conjugative plasmid and the T4SS encoded by the GGI

showed that both systems function independently in transfer of only plasmid or only

chromosomal markers, respectively .

The gonoccocal T4SS ene

sequences and the gene on

both systems revealed seve

from the relaxase of the F

TraM or TraY have been I

DNA transport and iv) the

the IncP incompatibility grc

the F plasmid and the T4S~

relatedness and functional

pair formation complex we:

involved in targeting the D

the coupling protein Tra[

islands, like the PAGI-2 an

PFGI-l genetic island of?

of the GGI is composed (

targeting components resel

previously shown that the!

the features of this novel fa

(DUF 1528). In this study

function of the TraD and

interact with the F plasmic

That again showed that eit

or more gonococcal compc

to be provided in trans to c

T4SSs playa crucial role i

has been made in identifyi

roles and substrates in pat

Data presented in this thes

understand the functional

pathogenesis. Secondly, tl

give more opportunities to

144

Chapter 5

were identified in a large

Dutch type gonoccoccal

I type backbone insertions

xt to the insertion of the

with the tetM detenn inant

5 MDa plasm ids) was the

the Dutch type backbone

for replication initiation,

inheritance and control.

rial conjugative plasmids

suggesting that they are

pe neisserial conjugative

PI plasm ids. Within this

Remarkably, two of the

family. Therefore, Dutch

of mUltiple copies of

the neisserial conjugative

ociated with virulence in

wn. In H. injluenzaea the

shown to function as an

a found next to the vapD

arkably, the Dutch type

gene. Interestingly, the

r cells strongly increased

coli, indicating that the

fapD on the conjugative

Ijugative plasm ids. Most

vantage of having three

; unclear. Comparison of

~S encoded by the GGI

. only plasmid or only

The gonoccocal T4SS encoded within the GGI shows strong similarity with the protein

sequences and the gene order of the F plasmid T4SS [5] . However, detailed comparison of

both systems revealed several differences: i) the relaxase TraI of the GGI differs strongly

from the relaxase of the F plasmid, ii) no accessory proteins for relaxosome formation like

TraM or TraY have been found, iii) no cell to cell contact is required for activation of the

DNA transport and iv) the pilin encoded in the GGI resembles the P-type pili found in e.g

the IncP incompatibility group and the Agrobacterium tumejaciens Ti plasm ids. In chapter 4

the F plasmid and the T4SS encoded within the GGI were compared in term of evolutionary

relatedness and functional conservation. This demonstrated that the proteins of the mating

pair formation complex were related to their homologs of the F plasmid, but that the proteins

involved in targeting the DNA to the mating pair formation complex, the relaxase TraI and

the coupling protein TraD cluster with proteins normally found in conjugative genetic

islands, like the PAGI-2 and pKLC102 genomic islands of Pseudomonas aeruginosa and the

PFGI-l genetic island of P. jluorescens Pf-5. This suggests that the type IV secretion system

of the GGI is composed of an F plasmid like mating pair formation system, but that the

targeting components resemble conjugative systems found in genetic islands. Indeed it was

previously shown that the gonoccocal TraI belongs to a novel family of relaxases [7] . One of

the features of this novel family of relaxases is the presence of a domain of unknown function

CDUF 1528). In this study we tested whether gonoccocal TraD and TraI may substitute the

function of the TraD and TraI deficient mutants of the F plasmid. Both proteins failed to

interact with the F plasmid self-transfer functions and for mobilization of the oriT plasmid.

That again showed that either both the F plasmid and the GGI function in a different manner

or more gonococcal components, possibly the coupling protein and the relaxase together need

to be provided in trans to complement the deletions.

T4SSs playa crucial role in bacterial pathogenesis. In the past few years remarkable progress

has been made in identifYing T4SSs of bacterial pathogens and in the understanding of their

roles and substrates in pathogenesis. However, many aspects remain to be still investigated.

Data presented in this thesis pose new challenges for future. One of the main goals will be to

understand the functional importance of the presence of a non mutated TraA in neisserial

pathogenesis. Secondly, the completed sequence of the neisserial conjugative plasmids will

give more opportunities to study this T4SS system in more details.

145