4 ACCEPTED - Journal of Bacteriology · 2007-09-14 · 14 terminal region of the TpsA proteins...

TITLE: 1

2

A functional Two-Partner Secretion System contributes to adhesion of Neisseria 3

meningitidis to epithelial cells 4

5

Running Title: A functional TPS in Neisseria meningitidis 6

7

AUTHORS: 8

9

Corinna Schmitt1, David Turner

2, Maria Boesl

1, Marion Abele

1, Matthias Frosch

1, Oliver 10

Kurzai1*

11

12

13

AFFILIATIONS 14

1; University of Wuerzburg, Institute of Hygiene and Microbiology, Josef-Schneider-Str. 2 / E1, 97080 15

Wuerzburg, Germany 16 2;

Institute of Infections, Immunity and Inflammation, School of Molecular Medical Sciences Centre for 17

Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK 18

19

20

21

22

23

Correspondent footnote: 24

25

Oliver Kurzai 26

Institute of Hygiene and Microbiology 27

University of Wuerzburg 28

Josef-Schneider-Str.2 29

97080 Wuerzburg 30

Phone: ++49 (0)931 20146905 31

Fax: ++49 (0)031 20146445 32

Email: [email protected] 33

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00851-07 JB Accepts, published online ahead of print on 14 September 2007

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Abstract 1

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Neisseria meningitidis is a frequent commensal of the human nasopharynx causing severe 3

invasive infections in rare cases. A functional two-partner secretion (TPS) system in N. 4

meningitidis, composed of the secreted effector protein HrpA and its cognate transporter 5

HrpB, is identified and characterized in this study. Although all meningococcal strains 6

harbour at least one TPS system, the hrpA genes display significant C-terminal sequence 7

variation. Meningococcal genes encoding the TPS effector proteins and their transporters are 8

closely associated and transcribed into a single mRNA. HrpA proteins are translocated across 9

the meningococcal outer membrane by their cognate transporters HrpB and mainly released 10

into the environment. During this process, HrpA is proteolytically processed to a mature 180 11

kDa form. In contrast to other known TPS systems, immature HrpA proteins are stable in the 12

absence of HrpB and accumulate within the bacterial cell. A small percentage of mature HrpA 13

remains associated with the bacteria and contributes to the interaction of meningococci with 14

epithelial cells. 15

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Introduction 1

2

Export of proteins to the surface and protein secretion are implicated in many aspects of 3

bacterial life, including cell to cell communication, motility and virulence. Highly 4

sophisticated systems have evolved to ensure correct secretion of bacterial proteins and many 5

of them have been recognized as central virulence determinants. These include the type IV 6

secretion system of Helicobacter pylori (2), type III secretion systems of Enterobacteriaceae 7

(32), and bacterial autotransporters (18). Filamentous hemagglutinin (FHA) of Bordetella 8

pertussis is the prototype of a family of large exoproteins in Gram-negative bacteria that are 9

secreted via the two partner secretion (TPS) pathway (22, 23). FHA and related proteins, 10

termed TpsA for two-partner-secretion-protein A, are translocated across the inner membrane 11

via the universal Sec machinery (8). Transport across the outer membrane requires a specific 12

transporter termed TpsB. In B. pertussis this transporter is referred to as FhaC (23). The N-13

terminal region of the TpsA proteins contains a signal peptide for secretion via Sec and the 14

adjacent TPS signal or secretion domain of approximately 245 aa that targets the TpsA 15

protein to its cognate transporter TpsB (8, 10, 22, 23). The genes encoding TpsA and TpsB 16

are often found in close vicinity. Several TpsA proteins have been shown to contribute to 17

virulence in plant and animal pathogens (5, 13, 38, 43, 48). B. pertussis FHA itself is both a 18

major adhesion essential for establishing pathogen – host contact (29) and an important 19

immunogen (26), representing the main constituent of the pertussis vaccine (35). By 20

subtractive hybridization of DNA regions specific for Neisseria meningitidis strain Z2491 and 21

absent in N. gonorrhoeae, Klee et al. were the first to describe a homologue of FHA in 22

meningococci (27). All three available meningococcal genomes contain ORFs encoding 23

putative two-partner secretion systems (4, 34, 44) (Table 1). According to their homology 24

with FHA the corresponding genes have been annotated as hemagglutinin/hemolysin-related 25

proteins in strain MC58 and will be referred to as HrpA (TpsA homologue) and HrpB (TpsB 26

homologue), respectively. In contrast to the other strains, the genome sequence of serogroup 27

B strain MC58 (44) contains two almost identical copies of the TPS system as well as three 28

additional putative hrpA genes which differ mainly in their C-terminal sequences (Table 1) 29

(46). In addition, another putative hrpB gene is present in MC58. The highest degree of 30

sequence similarity between B. pertussis FhaB and meningococcal HrpA exists within the N-31

terminal region of the predicted proteins (22-25% identity) comprising the domain necessary 32

for binding of FhaB to its transporter and secretion by the TPS pathway. In contrast, the C-33

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terminal sequences of the meningococcal HrpA proteins show no relevant homology to B. 1

pertussis FhaB. 2

N. meningitidis colonizes the upper respiratory tract of approximately 10% of healthy 3

individuals (6, 7, 12) and only occasionally invades the bloodstream causing life-threatening 4

septicaemia and meningitis (39). Whereas invasive meningococci belong to a few clonal 5

lineages in the vast majority of cases, isolates from healthy carriers are genetically highly 6

diverse (12). The genetic background of the variable virulence potential in N. meningitidis is 7

still unclear. However, a thorough comparison of the genetic composition of hyperinvasive 8

strains with carrier strains might lead to a better understanding of virulence in N. meningitidis. 9

By analysing a large collection of meningococcal carrier isolates we show that TPS systems 10

are present in all N. meningitidis strains. However, the effector proteins display a uniquely 11

high degree of sequence variation. By constructing deletion mutants, a function of HrpB as a 12

transporter for HrpA and the contribution of HrpA to meningococcal adherence to host 13

epithelial cells could be established. 14

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Materials and Methods 1

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Bacterial strains and culture conditions 3

4

A total of 822 N. meningitidis isolates obtained from healthy carriers were used for analysis, 5

which have been described previously (11, 12). Meningococcal strains used for infection 6

experiments are listed in supplementary Table 1. Strains were stored in a glycerol stock at –7

80°C and plated on GC-agar (BD Difco, Heidelberg, Germany) supplemented with Poly-8

ViteX (BioMerieux, Marcy l’Etoile, France). When appropriate, chloramphenicol (for N. 9

meningitidis 7µg/ml, for E. coli 30µg/ml), spectinomycin (125µg/ml, 75µg/ml), kanamycin 10

(100µg/ml, 30µg/ml) and/or erythromycin (7µg/ml) were added to the medium for selection. 11

12

13

Molecular cloning, plasmids, primer 14

15

Plasmids and primers are listed in supplementary Table 2. Recombinant DNA techniques 16

were performed according to standard laboratory procedures. Plasmids were introduced into 17

E. coli by chemical transformation. Meningococcal deletion mutants were constructed by 18

integrating a selection marker into the gene of interest, simultaneously deleting part of the 19

gene. Transformation of N. meningitidis was performed as described (15) and mutants were 20

selected on appropriate media (see above). Detailed information regarding mutant 21

construction can be found in Supplementary Methods. Meningococcal mutants used in this 22

study are listed in supplementary Table 1. All mutant strains were checked for LPS 23

immunotype (L3 and L8) in a standard ELISA using antibodies kindly provided by W. 24

Zollinger and expression of major surface markers (Opc, Opa, Pili). Capsule expression was 25

assayed using antibodies mAb 735 (serogroup B) and mAb924 (serogroup C) respectively 26

(14). mAb SM1 (anti class 1 pili) was kindly provided by M. Virji (Bristol, UK), mAb AD211 27

(anti class 2 pili), mAb B306 (anti Opc) and mAb 4B12/C11 (anti Opa) were kindly provided 28

by Mark Achtman (Berlin, Germany). 29

30

31

Chromosomal DNA isolation, PCR, Southern blot and DNA sequencing 32

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Chromosomal DNA was isolated from N. meningitidis using the Genomic-Tip system 1

(Qiagen, Hilden Germany). Restriction enzymes and Taq-Polymerase were purchased from 2

NEB (Frankfurt, Germany). For amplification of DNA-fragments larger than 4kb a high 3

fidelity Taq polymerase was used (Platinum Taq DNA polymerase high fidelity, Invitrogen). 4

Southern Blot analysis was performed as previously described (19). Automated DNA 5

sequencing was performed with the dye-deoxy terminator cycle method on an Applied 6

Biosystems 377 sequencer. 7

8

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Dot blot hybridization 10

11

Chromosomal DNA was prepared with the QIAmp DNA Mini Kit (Qiagen) and 200 ng 12

samples were spotted onto nylon membranes (Macherey-Nagel). Dot blot hybridization was 13

performed as described previously (19). Probes specific to the meningococcal hrpA genes 14

NMB0493, NMB1214 and NMB1768 were generated by PCR using the primers 15

OK99/OK100 (nucleotide 2050 to 4071), OK103/OK104 (nucleotide 2498 to 4526) and 16

OK105/OK106 (nucleotide 1999 to 4004) respectively. A single probe detecting both 17

NMB0497 and NMB1779 was generated by PCR using the primers OK90/OK91 (nucleotide 18

2482 to 4537). A probe specific to NMC0443 was amplified with the primers OK423/OK424 19

(nucleotide 752 to 1168). A probe specific for NMB1762 was amplified with primers 20

OK489/OK490 (nucleotide 2 to 1779). Probes were labelled using a random primer 21

digoxigenin system (Roche, Mannheim, Germany) and detected with the chemiluminiscence 22

detection kit (Roche). 23

24

25

RNA extraction and RT-PCR 26

27

RNA was prepared using RNeasy Kits (Qiagen) and reverse transcription PCR was performed 28

with the One-step RT-PCR Kit (Qiagen). To exclude DNA contamination of the samples, 29

PCR without RT was performed simultaneously. In strain MC58, expression of the hrpA 30

genes was analysed with primers OK301/OK302 (NMB0493), OK307/OK308 (NMB1768), 31

OK305/OK306 (NMB1214), and OK303/OK304 (NMB0497/NMB1779), each primer pair 32

amplifying internal fragments of the respective genes. In strain 2120, expression of the hrpA 33

gene NMC0444 was analysed with primers OK442/OK443. To analyse the expression of 34

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hrpB genes homologous to NMB1780 and to simultaneously verify the operon organization of 1

hrpA and hrpB genes, primer pairs were designed with the forward primer recognizing 2

sequences at the 3’ end of hrpB and the reverse primer recognizing a conserved region at the 3

5’ end of the associated hrpA gene. Strains were analysed with primers OK474/OK475 or 4

OK338/OK339. Expression of hrpB genes homologous to NMB1762 was analysed with 5

primers OK491/OK 492 (Primer sequences are listed in supplementary Table 2). 6

7

8

Expression in E. coli and purification of NMB1779 9

10

E. coli strain SCS1 containing pQE-NMB1779 (kindly provided by S. Klee) was grown 11

overnight in 10 ml of LB medium containing 100µg/ml ampicillin and 12µg/ml Kanamycin. 12

The following day, 1 ml of the overnight culture was used to inoculate 30 ml of fresh 13

medium. This culture was allowed to grow an OD600 of 0.5 – 0.6 before the addition of IPTG 14

to 0.5 mM. After 3 h, cells were harvested and the recombinant protein was affinity-purified 15

under denaturing conditions using Ni-NTA Spin Kit (Qiagen) according to the manufacturer’s 16

instructions. 17

18

19

Rabbit polyclonal antibodies against NMB1779 20

21

Rabbit polyclonal antibodies to NMB1779 (RαNMB1779) were raised against the denatured 22

affinity-purified recombinant protein in a New Zealand White female rabbit as previously 23

described (1). RαNMB1779 was used in immunoblots in a dilution of 1:100. 24

25

26

Western blot detection of HrpA proteins 27

28

Antibody RαNMB1779 was used to investigate the presence of HrpA proteins. For that 29

purpose, an overnight culture of N. meningitidis was inoculated into 20ml Dulbecco’s MEM 30

medium (Biochrom) supplemented with Poly-ViteX (BioMerieux) and grown for 1h at 31

37°C/200rpm. Cultures were adjusted to an OD600 of 0.1 in 20ml and grown until an OD600 of 32

1.2. For generation of concentrated supernatants, bacteria were pelleted by centrifugation and 33

the supernatant was filtrated through a 0.2 µm filter (Whatman). Supernatant proteins were 34

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concentrated with an Amicon Ultra-15 centrifugal filter device (Millipore). For generation of 1

whole cell lysates, cultures were adjusted to an OD600 of 0.6 in 1ml. Bacteria were harvested 2

by centrifugation and the resulting pellet was resuspended in 50µl of sample solution. 25 µl of 3

the respective samples were used for electrophoresis, blotted and incubated with the 4

respective antibody. Detection was performed with the SuperSignal West Pico 5

Chemiluminescent Substrate (Pierce). 6

7

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Infection experiments 9

10

HEp-2 epithelial cells were grown in RPMI1640 medium supplemented with 2mM L-11

glutamine and 10% heat inactivated fetal calf serum (FCS) in a 96-well microtiter plate until 12

confluent growth. FaDu epithelial cells (ATCC HTB-43) were grown in Eagle’s minimum 13

essential medium (Cambrex) with 2mM L-glutamine and Earle’s BSS adjusted to contain 1.5 14

g/L sodium bicarbonate, 0.1 mM non-essential amino acids (Cambrex), 1.0 mM sodium 15

pyruvate (Cambrex) and 10% heat inactivated FCS in a 96-well microtiter plate until 16

confluent growth. Prior to infection experiments, cells were washed with PBS and infection 17

experiments were performed in RPMI1640 medium without serum. Infection experiments 18

were performed as described previously (28). Cell-associated bacteria were calculated as 19

percent of total bacteria present in the well after 5h of infection. All experiments were 20

performed at least in triplicate unless otherwise indicated. Two-tailed Student`s t-test was 21

used to calculate significance values. 22

23

24

25

Protein and nucleic acid sequence analysis 26

27

Public databases of published protein and nucleotide sequences were searched using the 28

BLAST programs available at http://www.ncbi.nih.gov/BLAST/ and 29

http://tigrblast.tigr.org/cmr-blast/. The genome databases of meningococcal strain Z2491, 30

MC58 and FAM18 were searched using the BLAST servers available at TIGR 31

(http://www.tigr.org) and the Sanger Institute (http://www.sanger.ac.uk). Conserved domain 32

searches were performed using SwissPfam available at http://www.sanger.ac.uk, InterProScan 33

available at http://us.expasy.org/prosite/ and the NCBI Conserved Domain Search available at 34

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http://www.ncbi.nih.gov/. Protein sequences were aligned using the multiple sequence 1

alignment Clustal W available at http://www.ddbj.nig.ac.jp/search/clustalw-e.html. 2

Phylogenetic analysis was performed with MEGA (version 3.1.) available at 3

www.megasoftware.net. 4

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Results 1

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Presence and Distribution of hrpA genes among N. meningitidis carrier strains 4

A panel of 822 N. meningitidis isolates from healthy individuals was analysed for the 5

presence of hrpA genes. Genetic characteristics of these strains including MLST analysis have 6

been described previously (12). Presence and distribution of hrpA genes were analysed by 7

dot blotting with specific probes for NMB0493, NMB1214, NMB1768 and NMB0497/1779. 8

A total of 687 strains that gave unambiguous results were enrolled in the analysis 9

(supplementary Table 3). 97% (210/216) of the strains belonging to one of the known 10

hypervirulent lineages were found to be positive for at least one of the probes: 112/114 [98%] 11

isolates belonging to the ST-41/44 complex, 34/34 [100%] isolates belonging to the ST-32 12

complex, 59/63 [94%] belonging to the ST-23 complex, 3/3 [100%] isolates belonging to the 13

ST-11 complex and 2/2 [100%] isolates belonging to the ST-8 complex (supplementary Table 14

3). The majority of the remaining strains (334/471 [71%]) not belonging to hypervirulent 15

lineages also carried at least one hrpA gene. Among those strains that did not hybridize with 16

the probes, 113/137 [82%] belonged to four of five clonal complexes harboring the capsule 17

null locus (cnl) (11) including all strains of the ST-53 complex (52/52), the ST-198 complex 18

(42/42), the ST-1117 complex (17/17) and the ST-1136 complex (2/2). The majority of hrpA 19

positive strains (255/544 [47%]) hybridized only with the probe specific for NMB0497/1779 20

(supplementary Table 3). 159/544 [29%] strains hybridized with the probes specific to 21

NMB0493, NMB1768 and NMB0497/1779 but not with the probe for NMB1214. More than 22

50% of these strains were members of the ST-41/44 complex (86 isolates). A positive signal 23

with all four probes could be demonstrated in 76/544 strains [14%], including most isolates of 24

the ST-32 complex (33/34), of the ST-269 complex (12/12), and a set of strains belonging to 25

the ST-35 complex (10/32, 31%). Interestingly, the presence of a hrpA gene homologous to 26

either NMB0493, NMB1214 or NMB1768 was always associated with a positive signal for 27

NMB0497/1779. Though there were no isolates representing the three serogroup A related 28

hypervirulent lineages among the carrier strains analysed, the presence of one hrpA gene in 29

the published genome sequence of strain Z2491 suggests that hrpA are also distributed among 30

the serogroup A strains. 31

32

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Presence and Distribution of hrpB genes among N. meningitidis carrier strains 34

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Given the high sequence variability of known hrpA genes in N. meningitidis, strains that did 1

not react with any of the four probes could either completely lack a hrpA homologue or 2

contain hrpA genes not detected by the probes due to sequence variation. Therefore, 3

additional dot blots were performed with a probe specific to the highly conserved 4

meningococcal hrpB gene NMC0443 (Table 1). Almost all strains (801/822 [97.4 %]) 5

hybridized with the probe specific to the hrpB gene (Figure 1). As the genes for cognate 6

exoprotein and transporter protein are closely associated in known TPS systems, the presence 7

of genes coding for possible transporter proteins suggests that strains which were negative 8

with the MC58 hrpA probes harbored undetected hrpA genes differing in their nucleotide 9

sequence from those present in MC58. This hypothesis was tested by PCR of a selection of 30 10

strains representing hypervirulent clonal complexes (ST-8 complex, ST-11 complex, ST 23 11

complex, ST 41/44 complex) and non hypervirulent clonal complexes (ST-22 complex, ST-60 12

complex, ST-162 complex) including those harboring the capsule null locus (ST-53 complex, 13

ST-198 complex, ST-845 complex, ST-1117 complex). Additionally, the 21 strains which did 14

not hybridize with the hrpB probe were tested. The forward primer recognized the 3’-terminus 15

of the highly conserved hrpB gene (corresponding to nucleotide 1641 to 1663 in NMC0443). 16

The reverse primer recognized a conserved motif within the secretion domain present in all 17

known meningococcal hrpA genes (corresponding to ns 501 to 520 in NMC0444). A DNA 18

fragment with the expected size was amplified in all strains with the exception of one isolate 19

belonging to the ST-774 complex (data not shown). NMB1762 has been hypothesised to 20

encode the cognate transporter protein for HrpANMB1768 (46). By dot blot hybridization with a 21

probe specific to NMB1762, 328 out of the 822 strains analysed [40%] gave a positive result 22

(Figure 1). Almost all strains harboring NMB1768 homologous genes hybridized also with 23

the probe for NMB1762 (288/294 [97.9%]). In all six negative strains, PCR with primers 24

specific for NMB1762 revealed the presence of this gene. Similarly, the strains which 25

hybridized with the probe for NMB1762 but did not hybridize with the probe for NMB1768 26

or gave ambiguous results (34/328 [10.3%]) were shown to harbor both genes by PCR with 27

primers specific for NMB1768. The only exception was one isolate of the ST-167 complex 28

which was negative with both the probe as well as the PCR for NMB1768. 29

30

Sequence analysis of the hrpA gene from strain alpha 14 31

To determine whether strains that did not hybridize with the MC58 hrpA probes but displayed 32

a positive PCR result with the hrpA/hrpB primer pair harbor a full length hrpA gene, the 33

complete nucleotide sequence of the single hrpA gene of strain alpha 14 (cnl:ST-53) was 34

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determined from contigs of a whole genome sequencing project (Schoen et al., unpublished 1

data). The 5949 bp hrpAalpha14 (NM0_0398) encodes a protein with a predicted molecular 2

weight of approximately 207 kDa displaying an overall identity to NMB1779 of 50.8% with 3

the highest degree of similarity at the N-terminus (98.8 % identity within the first 321 amino 4

acids) including the TPS domain. Motifs critical for secretion of FHA in B. pertussis and 5

conserved in MC58 hrpA genes are also present in NM0_0398 (Supplementary Figure 1). In 6

contrast, the C-terminus was more diverse displaying only 41.5 % sequence identity, which 7

accounts for the failure of the MC58 derived probes to detect hrpAalpha 14. hrpAalpha 14 is most 8

closely related to NMC0444 with an overall nucleotide identity of 51.8% and an overall 9

amino acid identity of 78.6 %. A phylogenetic tree was constructed to compare HrpAalpha 14 10

with the other known meningococcal HrpA proteins. Accordingly, meningococcal HrpA 11

proteins can clearly be separated into two groups (Figure 2). HrpAalpha 14 clustered into group 12

I, comprising the single HrpA proteins of meningococcal strains Z2491 and FAM18 together 13

with the two HrpA proteins of strain MC58 encoded by NMB0497 and NMB1779, 14

respectively. Group II comprised the three HrpA proteins of strain MC58 encoded by 15

NMB0493, NMB1214 and NMB1768 which have no close homologues in the other two 16

available N. meningitidis sequences (Figure 2). Even HrpA proteins within one of these 17

groups display significant C-terminal sequence variation (Figure 2). The protein sequences 18

encoded by six putative hrpA genes identified in the preliminary whole genome sequence of 19

N.lactamica ST640 (available at www.sanger.ac.uk) were included in the analysis. 20

Exclusively genes encoding HrpA homologous to group II, but not to group I, meningococcal 21

HrpA proteins were detected in N. lactamica (Figure 2). 22

23

24

Expression analysis of hrpA and hrpB genes in a selection of meningococcal strains 25

Reverse transcriptase PCR (RT-PCR) was performed to detect the presence of hrpA and hrpB 26

transcripts in total RNA isolated from different meningococcal strains. In strain MC58, 27

expression was confirmed for the hrpA genes NMB0493, NMB1214, NMB1768 and 28

NMB0497/NMB1779 and for the hrpB genes NMB1780/NMB0496 and NMB1762. As 29

NMB0497 and NMB1779 and their respective associated putative transporters 30

NMB0496/NMB1780 display almost 100% sequence identity, differentiation between the 31

genes was not achieved. Using a forward primer recognizing the 3’ end of hrpB NMB1780 32

and a reverse primer recognizing a conserved region within the 5’-end of the hrpA gene 33

NMB1779 it could be shown that both genes are organised in an operon and transcribed into a 34

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single mRNA. Similar results were obtained with strain 2120 harboring only one hrpA gene 1

(Figure 3). Therefore the typical operon structure of other TPS systems is conserved in N. 2

meningitidis. The expression of hrpA genes was also analysed in a subset of the carrier 3

strains representing 11 clonal complexes using the hrpA/hrpB primer pair. In all strains, a 4

DNA fragment of the expected size was amplified (data not shown). Similarly, a selection of 5

9 carrier strains representing 9 different clonal complexes that had been shown to harbor 6

NMB1762 homologues could be shown to express the respective gene. Therefore, all hrpA 7

and hrpB genes were found to be transcribed in all tested meningococcal isolates. 8

9

10

Meningococcal HrpA proteins are expressed, proteolytically processed and secreted in 11

an HrpB dependent manner 12

To investigate the expression of meningococcal HrpA proteins, a polyclonal rabbit antiserum 13

was raised against the HrpA protein encoded by NMB1779. For that purpose, His-tagged 14

NMB1779 was expressed and purified in E. coli. The respective antiserum was able to 15

recognize recombinant HrpA protein in a Western blot which migrated at approximately 210 16

kDa (Figure 4). However, when Western blots were performed with whole cell lysates of 17

strain MC58, no band corresponding in size to the recombinant protein could be detected 18

(Figure 4). Instead, the antiserum did detect small amounts of a protein of approximately 180 19

kDa which was absent in ∆NMB0497 ∆NMB1779 mutants. Interestingly, analysis of a ∆hrpB 20

mutant (simultaneous deletion of NMB0496 and NMB1780) revealed a prominent band 21

corresponding in size to the full-length recombinant HrpA protein. Therefore, proteolytic 22

processing of meningococcal HrpA proteins might occur during or after translocation across 23

the outer membrane. To determine whether HrpA proteins are secreted into the environment, 24

whole cell lysates of strain 2517 and its respective ∆hrpA and ∆hrpB deletion mutants as well 25

as a ∆hrpA ∆hrpB double deletion mutant were compared with concentrated supernatants of 26

the same strains (Figure 4). Strain 2517 was chosen for these experiments as only one single 27

hrpA gene together with its associated hrpB gene are present in the latter strain. Similar to the 28

results obtained with the ∆hrpB mutant of strain MC58, a prominent band of approximately 29

210 kDa was detected in whole cell lysates of strain 2517 ∆hrpB (Figure 4). This protein 30

could not be detected in both the ∆hrpA and the ∆hrpA ∆hrpB mutant and most likely 31

represents the unprocessed HrpA protein accumulating in the bacterial cell due to the deletion 32

of its transporter HrpB. As for strain MC58, a band of approximately 180kDa was detected in 33

whole cell lysates of strain 2517. In addition, a protein of the same size was detected in the 34

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culture supernatants (Figure 4). This form of HrpA was absent from supernatants and cell 1

lysates of the ∆hrpA and the ∆hrpA ∆hrpB mutant. These data provide evidence, that HrpA 2

proteins are effectively released into the culture supernatant in the presence of HrpB. 3

Furthermore, HrpA proteins are processed during or after secretion as indicated by the lower 4

molecular mass of the mature HrpA. A small proportion of the processed HrpA remains 5

associated with the bacterial cell. 6

7

8

Contribution of hrpA to the interaction of meningococci with host cells 9

To investigate whether the presence of different hrpA genes in strain MC58 contributes to 10

adherence, mutants for NMB0493, NMB1214 and NMB1768 respectively were constructed 11

in the unencapsulated strain MC58 ∆siaD and the unencapsulated and LPS truncated strain 12

MC58 ∆siaD ∆lgtA by insertional inactivation of these genes. As NMB0497 represents an 13

almost identical copy of NMB1779, both genes were inactivated simultaneously in strain 14

MC58 ∆siaD and in the unencapsulated and LPS truncated strain MC58 ∆siaA-D ∆galE. 15

Inactivation of hrpA genes was confirmed by PCR and Southern Blot analysis (data not 16

shown). Unencapsulated and LPS-truncated parental strains were chosen because capsule 17

expression as well as the presence of the LPS alpha-chain shields meningococci from 18

interaction with epithelial cells (Kurzai et al, unpublished data). Infection experiments were 19

performed to quantify the adherence of the ∆hrpA mutants to epithelial cells in comparison to 20

their respective parental strains. The proportion of cell-associated bacteria (adherent and 21

viable intracellular) was quantified 5 h p.i. No difference could be detected in the proportion 22

of cell-associated bacteria between the hrpA mutants and their parental strains (data not 23

shown). These data indicate that adherence to epithelial cells is not mediated by an individual 24

hrpA present in MC58. As it can not be excluded that the proteins encoded by the different 25

hrpA genes share a common function in the interaction with host cells, further infection 26

experiments were performed using mutants of serogroup C strain 2120 harboring only a 27

single hrpA gene. HrpA mutants were constructed in the unencapsulated strain 2120 ∆siaD 28

and in the unencapsulated and LPS truncated strain 2120 ∆siaD ∆lgtA by insertional 29

inactivation. Irrespective of the parental strain genotype, deletion of hrpA or hrpB did not 30

influence growth kinetics (data not shown). Whereas no difference in the number of HEp-2 31

cell-associated bacteria could be detected for strain 2120 ∆siaD and its respective hrpA 32

mutant, deletion of the hrpA gene in the LPS truncated strain led to a significant decrease in 33

the proportion of cell-associated bacteria 5h p.i. [16.8% vs. 34.4% cell-associated bacteria; 34

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p<0.01] (Figure 5). Similar results were obtained with FaDu epithelial cells (Figure 5), 1

indicating that HrpA contribute to meningococcal adherence to different epithelial cell lines in 2

an unencapsulated and LPS-truncated background and might be functionally redundant in 3

strains carrying more than one allele. The same results were obtained with three additional 4

independent hrpA mutants of strain 2120 ∆siaD ∆lgtA (data not shown). Additionally, 5

deletion of the hrpB gene in strain 2120 ∆siaD ∆lgtA also led to a significant decrease in the 6

proportion of cell-associated bacteria 5 h p.i. (Figure 5) thus providing further evidence for 7

the functional relevance of HrpA secretion by its associated HrpB. In contrast to epithelial 8

cells, no difference could be detected in the number of cell-associated bacteria in infection 9

experiments with human dendritic cells (data not shown) suggesting that the adhesive 10

properties of HrpA are cell type specific. 11

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Discussion 1

2

Several large exoproteins in various gram-negative bacteria are predicted to be secreted via 3

the Two-partner secretion (TPS) system (23). However, relatively few of these proteins have 4

been investigated in detail. FHA, the most extensively characterized of these exoproteins, is 5

an important virulence factor of Bordetella and constitutes the main component of the 6

pertussis vaccine (29). In this study, meningococcal hrpA genes encoding high-molecular-7

weight proteins related to FHA were analysed. The sequence similarities between FHA and 8

HrpA are restricted to the N-terminus comprising the TPS domain which is the hallmark of 9

proteins secreted by the two-partner secretion pathway. In contrast, the C-terminal parts of the 10

proteins that have been shown to contain the functional domains in Bordetella FHA (31) show 11

no relevant homology between both species. The conserved TPS domain contains secretion 12

determinants mediating translocation of the respective protein across the outer membrane by 13

its associated transporter termed FhaC in B. pertussis (21, 23, 46). The presence of the TPS 14

domain including conserved NPNGI and NPNL motifs in the derived protein sequence of the 15

hrpA genes of strains MC58, Z2491, FAM18 and alpha 14 together with the fact that more 16

than 99% of the strains investigated in this study contain fhaC homologous genes suggests the 17

presence of a functional TPS in N. meningitidis. 18

Klee et al. were able to demonstrate the presence of genes homologous to the hrpA gene of 19

strain Z2491 in 13 strains mainly representing the known hypervirulent lineages of N. 20

meningitidis (27). In this study, by analysing a large collection of carrier isolates comprising 21

51 clonal complexes, we provide evidence that hrpA genes are ubiquitously present in N. 22

meningitidis and not restricted to strains of the hypervirulent lineages. According to their 23

degree of sequence similarity we propose to divide the meningococcal hrpA genes into two 24

groups. Group I comprises the two almost identical genes NMB0497 and NMB1779 together 25

with the single hrpA genes present in strain Z2491 (NMA0688), strain FAM18 (NMC0444) 26

and strain alpha 14 (NM0_0398). Group II comprises NMB1768, NMB0493 and NMB1214. 27

As no genes closely related to the latter group had until now been identified in meningococcal 28

strains other than MC58, our data for the first time provide evidence that they are not 29

restricted to MC58 or its close relatives. From an evolutionary point of view it is interesting to 30

notice that exclusively hrpA genes homologous to group II were detected in N. lactamica 31

strain ST640. These findings are congruent with a recent study by Snyder et al. (41). The 32

authors were able to demonstrate that genes homologous to NMB0493 and NMB1214 are 33

present in all 13 N. lactamica strains investigated using comparative genome hybridization to 34

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a pan-Neisseria microarray (41). Genes homologous to NMB1768 were detected in a subset 1

of N. lactamica strains whereas genes homologous to NMB1779 could not be detected in N. 2

lactamica (41). Similarly, the genome of N.gonorrhoeae strain FA 1090 contains an ORF 3

disrupted by multiple stop codons, which otherwise would encode a TpsA protein with 4

homology to NMB1768 (46). Even hrpA genes from the same group can show considerable 5

sequence variation in the C-terminus, which might be due to a direct recombination based 6

mechanism for C-terminus exchange between hrpA genes and a number of silent cassettes 7

encoding alternative C-termini present in the meningococcal genome (4, 46). For example, 8

NMB1779 and hrpAalpha 14 which both belong to group I meningococcal hrpA genes show a 9

sequence diversity of >49%. It is intriguing to speculate that C-terminal sequence variation 10

might have arisen from selective pressure and could alter the functions of these proteins and 11

modulate the virulence potential of different clonal lineages. 12

HrpA proteins are expressed and released into the culture supernatant by a HrpB dependent 13

mechanism. However, in contrast to B. pertussis and other known TPS systems, where the 14

exoprotein FHA is rapidly degraded intracellularly in the absence of the transporter FhaC (21) 15

(9, 25), meningococcal HrpA accumulates in the bacteria and remains stable and undegraded 16

if HrpB has been deleted. Our data strongly suggest that the HrpA protein is proteolytically 17

processed during or after secretion. A similar processing has been described for B. pertussis 18

(29) and H . influenzae (3). Details of the secretion and processing of HrpA will have to be 19

addressed in further studies. 20

Until now, the role of FHA related proteins in the interaction with the human host has only 21

been studied in detail in a few bacterial species. B. pertussis FHA mediates adherence to 22

epithelial cells (20, 37, 45) and macrophages (36, 40) and is required for tracheal colonization 23

in vivo (26). Similarly, the FHA-related proteins of nontypeable H. influenzae, HMW1 and 24

HMW2, were shown to mediate binding to macrophages (33) and human epithelial cells (42). 25

Our data clearly show that meningococcal HrpA proteins contribute to adherence to host cells. 26

A hrpA deletion mutant of strain 2120, which harbors only one hrpA gene, displayed 27

significantly reduced adherence to different epithelial cells. This effect was observed in an 28

unencapsulated background with short LPS (L8 immunotype). It has previously been shown 29

that capsule expression as well as expression of the LPS alpha-chain can shield meningococci 30

from interaction with host cells (28). Both capsule expression and expression of the LPS 31

alpha-chain is subject to phase variation in meningococci (17, 24) and therefore capsule 32

deficient as well as LPS truncated variants may play a role during colonization and 33

pathogenesis (17, 30). Indeed, the function of Opc, a well known adhesin and invasin of N. 34

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meningitidis is also dependent on a lack of capsule expression and LPS truncation (47). Due 1

to the fact, that only low amounts of HrpA remain cell associated and can be detected in 2

whole cell lysates, it cannot be excluded, that the effect of hrpA / hrpB deletion on adhesion is 3

indirect e.g. by altering outer membrane composition. However, no gross changes in 4

expression of outer membrane proteins could be detected in the deletion mutants. The role of 5

hrpA genes with homology to NMB1768, NMB0493 and NMB1214 has not been addressed 6

selectively as strains harboring one of these genes always possess additional hrpA genes 7

homologous to NMB1779/NMB0497. In strain MC58, deletions of single hrpA had no effect 8

on adhesion, and although we were able to construct a double deletion of NMB1779 and 9

NMB0497 in MC58, the mutant displayed the same adherence phenotype as the parental 10

strain. This might indicate that hrpA of both groups are functionally redundant in interaction 11

with epithelial cells despite considerable sequence difference. 12

In summary, FHA-like proteins (HrpA) as well as FhaC-like proteins (HrpB) are encoded and 13

expressed by virtually all N. meningitidis strains belonging to both hypervirulent and non-14

hypervirulent clonal complexes. HrpA proteins are secreted via transporters encoded by hrpB 15

genes and accumulate in the bacteria after deletion of this transporter. However, Western blot 16

analysis as well as the observation that HrpA proteins play a role in adhesion to host cells 17

indicates that a proportion of the secreted HrpA remains surface-associated. A similar 18

phenomenon has also been described with another meningococcal adhesin of the 19

autotransporter family, App (16). By analogy to the situation in B. pertussis, this renders these 20

proteins interesting candidates for designing potential vaccines against serogroup B 21

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Acknowledgements 1

2

This work would not have been possible without the excellent technical assistance of Julia 3

Oechsner. Antibodies have been kindly donated by Wendell Zollinger, Sanjay Ram, Mark 4

Achtman and Mumtaz Virji. The NMB1779 expression construct was kindly donated by Silke 5

Klee. Heike Claus and Ulrich Vogel contributed to this work in many helpful discussions and 6

provided dot-blots of the strains from the Bavarian carriage study. Christoph Schoen kindly 7

provided data from the alpha14 genome project. This work was supported by the German 8

Research Foundation (DFG) SFB479 (grant B2 to O.K. and M.F.). 9

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Table 1: hrpA and hrpB genes in the sequenced meningococcal genomes 1

2

hrpA hrpB

Strain ORF Predicted

protein

length (aa)

% identity

(protein) to

NMB1779

ORF Predicted

protein

length (aa)

% identity

(protein) to

NMB1780

Z2491 NMA0688 2016 77,4 NMA0687 580 98,6

NMB1779

1995 100 NMB1780

580 100

NMB0497

1976 93,5 NMB0496 1)

559 99,3

NMB0493

2703 13,4 --2)

-- --

NMB1214

2274 16,0 --2)

-- --

MC58

NMB1768 2514 13,9 NMB1762 595 44,5

FAM18 NMC0444 2027 65,4 NMC0443 580 99,0

3

4

NMA, NMB and NMC denote the genes in Z2491, MC58 and FAM18 genomes respectively 5

(44) encoding proteins homologous to B. pertussis FhaB (in meningococci termed HrpA) and 6

FhaC (in meningococci termed HrpB). 1)

The protein encoded by NMB0496 lacks the first 21 7

amino acids at the N terminus and thus presumably contains no functional signal sequence. 2)

8

NMB0493 und NMB1214 seem to encode singular HrpA as no associated putative hrpB gene 9

could be detected in the MC58 genome sequence. 10 ACCEPTED

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Figure Legends 1

2

Figure 1 3

DNA dot blot analysis of a selection of carrier strains with probes for the hrpA genes 4

NMB0493 (A), NMB1214 (B), NMB1768 (C) and NMB0497/1779 (D) as well as the hrpB 5

genes NMB1762 (E) and NMB0496/1780 (F). Each blot (A-F) shows the results of a 6

selection of 100 carrier isolates (alpha 201 to 300). The order of bacterial strains is the same 7

in all panels. The figures below each panel denote the percentage of strains out of the 822 8

strains analyzed that hybridized with the respective probes. The location of the probes used 9

for hybridization is indicated by black bars above the open rectangles representing the 10

respective hrpA and hrpB ORFs. The numbers below the open rectangles indicate the sizes (in 11

kilobases) of the ORFs. 12

13

Figure 2 14

Phylogenetic tree based on the deduced amino acid sequences of the proteins encoded by the 15

hrpA genes of strain MC58, Z2491, FAM18 and alpha 14 respectively. In addition, the 16

protein sequences encoded by six putative hrpA genes present in the preliminary sequence of 17

Neisseria lactamica ST640 were included in the analysis. The meningococcal HrpA proteins 18

clearly cluster into two separate groups. The HrpA identified in strain alpha 14 (denoted by 19

NM0_0398 in the figure) is closely related to the group of HrpA proteins encoded by genes 20

homologous to NMB1779. In contrast, the six HrpA identified in N. lactamica (denoted by 21

NLA8870, NLA12910, NLA10070, NLA8290, NLA8610 and NLA8550 in the figure) are 22

closely related to the second group of meningococcal HrpA proteins encoded by NMB0493, 23

NMB1214 and NMB1768. BP1879 denotes the ORF encoding the Filamentous 24

hemagglutinin of B.pertussis. Alignments were performed with ClustalW. Tree construction 25

was performed with Mega (version 3.1.) using the neighbour-joining method. Regions of the 26

alignment containing gaps were excluded from the analysis. 27

28

Figure 3 29

RT-PCR analysis of hrpA and hrpB expression in strain 2120. (A) Schematic representation 30

of the primer pairs used in (B). (B) Transcripts of hrpA and hrpB were detected in total RNA 31

preparations of strain 2120. Additionally, it could be shown that both genes are transcribed 32

into a single mRNA. The following templates were included in the samples: lane 1-3, lane 5, 33

lane 6-9: total RNA of strain 2120; lanes 4 and 9: no template (negative control); lane 10: 34

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27

genomic DNA of strain 2120 (positive control). The primer sets included in the samples were 1

as followed: lanes 1 and 6: hrpB specific primers (OK500/501, see A); lanes 2, 7 and 10: 2

hrpA specific primers (OK442/443, see A); lanes 3 and 8: primer pair with the forward primer 3

recognizing the 3’ end of the hrpB gene and the reverse primer recognizing a conserved 4

region within the 5’-end of the adjacent hrpA (OK338/339, see A) lanes 4, 5, 8 and 9: primers 5

specific for lgtA (OK487/488, positive control). The samples loaded in lanes 6 to 10 were not 6

subjected to the reverse transcription step of the RT-PCR procedure and served as controls to 7

rule out DNA contamination of the RNA templates. 8

9

10

Figure 4 11

12

Expression analysis of HrpA proteins by Western immunoblot assays. Proteins were separated 13

on a 7.5 % polyacrylamide gel, transferred to a nitrocellulose membrane and probed with 14

RαNMB1779. The arrows indicate the processed (white arrow) and unprocessed (black 15

arrow) HrpA proteins respectively. (A) Western blot of whole cell lysates of strain MC58, 16

strain 2517 and their respective ∆hrpA and ∆hrpB mutants. Lanes:1, purified HrpA; 2, MC58 17

wild type; 3, MC58 ∆siaD ∆hrpA; 4, MC58 ∆galE ∆hrpA; 5, MC58 ∆siaD ∆lgtA ∆hrpB; 6, 18

2517; 7, 2517 ∆hrpA; 8, 2517 ∆hrpB. In strain MC58, ∆hrpA denotes simultaneous deletion 19

of NMB0497 and NMB1779 whereas ∆hrpB denotes simultaneous deletion of NMB0496 and 20

NMB1780. M, molecular weight standard. (B) Coomassie stained gel of whole cell lysates 21

used in (A). (C) Western blot of culture supernatants of strain 2517 and its respective ∆hrpA 22

and ∆hrpB deletion mutants as well as the double deletion mutant ∆hrpA ∆hrpB in 23

comparison to whole cell lysate of strain 2517 ∆hrpB. Lanes: 1, 2517; 2, 2517 ∆hrpA; 3, 2517 24

∆hrpB; 4, 2517 ∆hrpA ∆hrpB; 5, whole cell lysate of strain 2517 ∆hrpB; 6, 2517. (D) 25

Coomassie stained gel of culture supernatants used in (C). 26

27

Figure 5 28

29

Adhesion of a ∆hrpA and a ∆hrpB deletion mutant of strain 2517 ∆lgtA to HEp2 (A) and 30

FaDu epithelial cells (B) 5h p.i. In comparison to the parental strain the proportion of cell-31

associated bacteria was significantly lower in the ∆hrpA mutant as well as in the ∆hrpB 32

mutant. Means and standard deviation of ten independent experiments are indicated. Two-33

tailed Student`s t-test was used to calculate significance values. 34

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1

2

3

4

5

6

7

8

9

10

32% 15% 36% 63% 40% 99%

A B C D E F

8.112 kb 6.836 kb 7.545 kb 1.78 kb 1.743 kb5.988 kb

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0.1

BP1879

NMB0493

NMB1768

NMB1214

NMC0444NM0_0398 NMA0688

NMB0497

NMB1779

0.1

NLA10070

NLA12910

NLA8870

NLA8550

NLA8290NLA8610

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A

OK 500/501

OK 338/339

OK 442/443

hrpB hrpA

B 1 2 3 4 5 6 7 8 9 10

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A 200 kDa

1 2 3 4 5 6 7 8M

B175 kDa

83 kDa

1 2 3 4 5 6 7 8M

1 2 3 4

C 175 kDa

5 6

175 kDa

D83 kDa

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A

Cell-associated

bacteria

[in % of total]

50

40

30

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0

p<0,01

p<0,01

0

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50

1

wt ∆hrpA ∆hrpB

B

50

40

30

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0

p<0,01

p<0,05

wt ∆hrpA ∆hrpB

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4 ACCEPTED - Journal of Bacteriology · 2007-09-14 · 14 terminal region of the TpsA proteins...

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