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J Bacteriol. Jul 2007; 189(14): 5399–5402.
Published online May 11, 2007. doi:  10.1128/JB.00300-07
PMCID: PMC1951848

Two ABC Transporter Operons and the Antimicrobial Resistance Gene mtrF Are pilT Responsive in Neisseria gonorrhoeae[down-pointing small open triangle]

Abstract

Retraction of type IV pili is mediated by PilT. We show that loss of pilT function leads to upregulation of mtrF (multiple transferable resistance) and two operons encoding putative ABC transporters in Neisseria gonorrhoeae MS11. This effect occurs indirectly through the transcriptional regulator FarR, which until now has been shown to regulate only farAB. l-Glutamine can reverse pilT downregulation of the ABC transporter operons and mtrF.

Type IV pili (TFP) are found in many gram-negative bacteria, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae (17). In addition to their role in DNA uptake (5), they also mediate twitching motility (17, 36). Recently, TFP were shown to be required for twitching motility not only in gram-negative bacteria but also in the gram-positive pathogen Clostridium perfringens and other clostridia (34).

Neisseria TFP play an important role in infection, mediating the initial attachment of bacteria to host cells (9, 21, 24). Retraction of the N. gonorrhoeae TFP triggers various epithelial responses, such as cortical plaque formation and initiation of signal cascades, and regulates host cell gene expression (2, 11, 15, 18, 20). The retraction force of a single TFP filament reaches 100 pN (16, 22). These and other findings indicate that TFP retraction is an important means by which N. gonorrhoeae communicates with the epithelial cell.

TFP retraction is dependent on PilT (22), which belongs to a highly conserved family of ATPases with homology to AAA-type motor proteins (19, 33). For several members of this family, ATP hydrolysis has been demonstrated in vitro (27, 29). The structure of the gonococcal PilT is similar to type II and type IV secretion ATPases in overall shape, size, and assembly (10). A pilT mutant of N. gonorrhoeae MS11 is nonmotile, hyperpiliated, and noncompetent for DNA uptake but adheres to human epithelial cells like the wild-type (wt) parent strain (15, 37). Based on a number of findings, PilT is proposed to cause TFP to retract by disassembling pilins at the base of the fiber (23).

Upregulation of putative ABC transporter genes and mtrF in the MS11 pilT mutant.

By comparing transcriptomes of N. gonorrhoeae MS11 and an otherwise isogenic pilT-derivative, MS11 pilT (15, 37), during adhesion to T84 human colorectal epithelial cells, we identified several genes encoding putative transporter proteins that are two- to threefold upregulated, with a P value of <0.05 in the pilT mutant (data from microarray experiments; not shown). The following open reading frame numbers correspond to the complete genomic sequence of N. gonorrhoeae FA1090, which is available under accession number AE004969. The identified pilT-responsive putative transporter genes belong to two sets. The gene products of the clusters NGO0372 to NGO0374 and NGO2011 to NGO2014 have highest homology to bacterial transporters that import amino acids, and NGO1368 encodes the protein MtrF (multiple transferable resistance) that is required for constitutive high-level resistance to hydrophobic agents through the MtrCDE efflux pump (35). To confirm upregulation of these transporter genes in the pilT mutant, relative quantification of gene transcription in wt MS11 and the pilT mutant, MS11ΔpilT, was performed by real-time quantitative reverse transcription (RT)-PCR. The MS11ΔpilT strain contains a 504-bp in-frame deletion in pilT. As for the microarray experiments, T84 epithelial cells were grown to 80% confluence and infected at a multiplicity of infection of 100. Infected cells were washed with phosphate-buffered saline to remove nonadherent bacteria, and subsequently, eukaryotic and bacterial RNA was purified (QIAGEN). For microarray experiments bacterial RNA depleted of eukaryotic RNA (MicrobEnrich; Ambion) was used to generate comparable amounts of labeled bacterial cDNA, while for real-time quantitative RT-PCR the Ambion procedure to deplete eukaryotic RNA was omitted. Instead, the purified RNA was directly used to generate cDNA. Real-time PCR using SYBR Green PCR master mix was carried out on an ABI Prism 7000 sequence detector system (Applied Biosystems). Our data show that a mutation in pilT leads to an approximately twofold upregulation (P values of <0.001) of the putative ABC transporter genes and mtrF, implicating PilT in the regulation of these genes (Fig. (Fig.11).

FIG. 1.
Expression of putative ABC transporter genes (NGO0372, NGO2011, and NGO2014), mtrF, and farR in strain MS11ΔpilT 4 h postinfection. Transcript levels were measured by real-time quantitative RT-PCR. Each column represents the relative change of ...

Effect of pilE on mtrF and the ABC transporter genes.

PilT may bring about these transcriptional changes through its function in TFP retraction or through a second unknown activity. To rule out the latter possibility, we compared the transcription of mtrF and the putative ABC transporter gene clusters (NGO0372-NGO0374 and NGO2011-NGO2014) in wt MS11 and MS11-307. MS11-307 is a mutant strain deleted of both pilin expression sites and does not produce TFP. It grows normally in liquid and agar media but adheres 6 logs less well to epithelial cells (1). We reasoned that if the transcriptional responses to pilT are due to its TFP retraction function, the same responses would also be seen in MS11-307. As in the pilT mutant, mtrF and the ABC transporter genes were upregulated ~2.5-fold (P values of <0.001) in MS11-307 (Fig. (Fig.2).2). This suggests that N. gonorrhoeae responds to a dysfunction in TFP (pilE and pilT) with an upregulation of mtrF and the ABC transporter gene clusters NGO0372-NGO0374 and NGO2011-NGO2014.

FIG. 2.
Expression of putative ABC transporter genes (NGO0372 and NGO2011) and mtrF in the pilT mutant, the MS11ΔpilT strain, and the pilE mutant, MS11-307. Transcript levels were measured by real-time quantitative RT-PCR. Each column represents the relative ...

Effect of l-glutamine on ABC transporter and mtrF transcription.

The pilT-responsive putative operons NGO0372-NGO0374 and NGO2011-NGO2013 encode proteins with homology to ABC transporters that are involved in amino acid uptake in bacteria (25). All the components of such importers—the ATP binding protein, a periplasmic binding protein, and the permease—are encoded within NGO0372-NGO0374. The putative operon NGO2011-NGO2013 encodes two permeases and one ATP binding protein. NGO2014, which is located 113 bp downstream of the tightly clustered NGO2011-NGO2013 genes, encodes the periplasmic amino acid binding protein of this putative transporter. NGO0372 shows highest similarities to periplasmic amino acid binding proteins that bind glutamine (26, 32). To test whether l-glutamine influences transcription of the putative ABC transporter genes and mtrF, MS11 was grown in cell culture dishes with GCB medium supplemented with glucose and cocarboxylase but without l-glutamine. After 3 h, l-glutamine was added to a final concentration of 2 mM, and the bacteria were incubated for one additional hour. The NGO0372, NGO2011, and mtrF transcripts from l-glutamine-supplemented cultures were compared to those from a culture without l-glutamine. The presence of l-glutamine resulted in a two- to threefold upregulation of NGO0372, NGO2011, and mtrF in MS11 with a P value of <0.0001 (Table (Table1).1). This response was not due to a general, nonspecific effect of l-glutamine on gene expression, as the pilF transcript, serving as the internal control, was unaffected. (The pilT mutation does not affect pilF transcription. [A. Friedrich, unpublished data]). Supplementation with amino acid l-arginine or l-asparagine had no effect on NGO0372, NGO2011, and mtrF (Table (Table1),1), indicating that the upregulation was specific to l-glutamine.

TABLE 1.
Influence of l-glutamine on the transcription of the ABC transporter genes (NGO0372 and NGO2011) and mtrF in MS11a

Several lines of evidence suggest that the pilT-responsive putative ABC transporter gene clusters NGO0372-NGO0374 and NGO2011-NGO2014 transport glutamine. The expression of many high-affinity importer genes is known to be induced by their substrate (3, 28). Consistent with this is our finding that l-glutamine upregulates transcription of these ABC transporter genes (Table (Table1).1). Studies have linked glutamine transport to virulence in other bacterial pathogens. Klose and Mekalanos showed that blocking glutamine synthesis and high-affinity transport attenuates virulence in Salmonella enterica serovar Typhimurium (12). The glutamine transport gene, glnQ, in group B streptococci is required for adherence to fibronectin and virulence in vivo (31). In this context, it is worth noting that NGO0372 and its N. meningitidis homologue, NMB0787, are upregulated upon adherence of wt Neisseria to host cells (6, 8). Gene disruption studies strongly suggest that NGO0372 is essential for viability of N. gonorrhoeae (7). Further studies will be necessary to determine whether the ABC transporter protein(s) functions in virulence.

In contrast to its effect in MS11, l-glutamine had little to no effect on NGO0372, NGO2011, and mtrF expression in MS11ΔpilT. In MS11ΔpilT, l-glutamine increased gene expression 1.1- to 1.3-fold (±0.2) over the no-glutamine control (data not shown). This suggests that TFP retraction plays a role in this regulation. How TFP retraction might bring this about is unclear. PilT, and perhaps also other components of the TFP biogenesis machinery, could be sensing glutamine concentrations in the extracellular environment. Alternately, TFP could be directly involved in glutamine import. It was shown that heme uptake in N. gonorrhoeae is dependent on the PilQ pore and PilT (4), which suggests a link between TFP biogenesis or TFP retraction and uptake of extracellular compounds.

It is tempting to speculate that MtrF might be also involved in glutamine uptake, as its expression is also upregulated by l-glutamine (Table (Table1).1). MtrF works through the MtrCDE efflux pump to confer high-level constitutive resistance to hydrophobic agents (35), but its exact mechanism of action is unknown. In terms of virulence, hybridization of genomic DNA from several Neisseria strains to the pan-Neisseria array (J. K. Davies et al., unpublished data) revealed that mtrF is present in pathogenic members of the Neisseriaceae family, N. gonorrhoeae and N. meningitidis, but not in commensal Neisseria (30).

Influence of FarR on pilT regulation of mtrF and ABC transporter genes.

FarR belongs to the regulatory MarR family and represses transcription of the farAB operon (13), which mediates resistance to antibacterial long-chain fatty acids. farR (fatty acid resistance) transcription is repressed by MtrR, which also regulates the mtrCDE operon and mtrF (14). Interestingly, farR was found to be twofold (± 0.05) downregulated in the pilT mutant during infection (with a P value of <0.05) (microarray data; not shown). Real-time quantitative RT-PCR was performed on wt- and MS11ΔpilT-infected cells to validate this result (Fig. (Fig.1).1). The farR transcript was consistently 1.6-fold (± 0.1) lower in MS11ΔpilT than in MS11 (P value of <0.001), indicating that in a wt pilT background, farR expression is normally upregulated. Although a 1.6-fold change in transcription level is relatively small, in the case of a repressor, this change could significantly alter gene expression.

To determine if pilT affects transcription of the putative ABC transporter gene clusters (NGO0372-NGO0374 and NGO2011-NGO2014) through FarR, a nonpolar mutation in farR was created in the MS11 background to generate the MS11 farR strain. MS11, MS11ΔpilT, and MS11 farR were grown in the presence of epithelial cells for 4 h, and the mtrF and ABC transporter transcripts (NGO0372-NGO0374 and NGO2011-NGO2014) were measured by real-time quantitative RT-PCR (Fig. (Fig.3).3). NGO0372 and mtrF were upregulated in response to the loss of farR (Fig. (Fig.3),3), suggesting that FarR normally represses their expression. However, the extent of upregulation in the MS11 farR strain was noticeably less than in MS11ΔpilT (1.8- to 2-fold and 2.5- to 3-fold, respectively). One possible explanation for this difference in response is that pilT normally upregulates farR expression. The loss of pilT would reduce farR expression, and this in turn would result in higher levels of NGO0372-NGO0374 and mtrF transcription. Alternatively, the higher level of NGO0372-NGO0374 and mtrF upregulation in the pilT mutant (Fig. (Fig.3)3) may reflect the involvement of an additional factor. In contrast to NGO0372, transcription of NGO2011 does not seem to be significantly influenced by a mutation in farR (Fig. (Fig.3).3). However, regulation of NGO2011 by FarR under different conditions than tested cannot be excluded.

FIG. 3.
Expression of mtrF and putative ABC transporter genes (NGO0372 and NGO2011) in the MS11 ΔpilT and MS11 farR strains. Transcript levels are measured by real-time quantitative RT-PCR 4 h postinfection. Each column represents the relative change ...

Taken together, our results suggest a regulatory network linking pilT and farR to the regulation of several transporter genes. Furthermore, our data indicate that the pilus retraction protein PilT might be required for the bacterial response to environmental signals, such as the availability of extracellular l-glutamine.

Acknowledgments

This work was supported in part by National Institutes of Health grant RO1-AI049973 awarded to M.S. Work from the Shafer laboratory was supported by National Institutes of Health grant AI-21150 and funds from a VA Merit Award. W.S. is the recipient of a Senior Research Career Scientist Award from the VA Medical Research Service. A.F. was supported by a fellowship within the Postdoc-Programme of the German Academic Exchange Service.

We thank Heather L. Howie for help and advice in the performance and analysis of microarray experiments. We thank Shelly Shiflett (Oregon Health Sciences University, Portland, OR), Nathan Weyand (Oregon Health Sciences University, Portland, OR), and Mirjana Kessler (MPI, Berlin, Germany) for critically reading the manuscript. We thank Thomas F. Meyer for fruitful discussion.

Footnotes

[down-pointing small open triangle]Published ahead of print on 11 May 2007.

REFERENCES

1. Arvidson, C. G., R. Kirkpatrick, M. T. Witkamp, J. A. Larson, C. A. Schipper, L. S. Waldbeser, P. O'Gaora, M. Cooper, and M. So. 1999. Neisseria gonorrhoeae mutants altered in toxicity to human fallopian tubes and molecular characterization of the genetic locus involved. Infect. Immun. 67:643-652. [PMC free article] [PubMed]
2. Ayala, P., J. S. Wilbur, L. M. Wetzler, J. A. Tainer, A. Snyder, and M. So. 2005. The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca(2+) transients in infected epithelial cells. Cell Microbiol. 7:1736-1748. [PubMed]
3. Belyaeva, T. A., J. T. Wade, C. L. Webster, V. J. Howard, M. S. Thomas, E. I. Hyde, and S. J. Busby. 2000. Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein. Mol. Microbiol. 36:211-222. [PubMed]
4. Chen, C. J., D. M. Tobiason, C. E. Thomas, W. M. Shafer, H. S. Seifert, and P. F. Sparling. 2004. A mutant form of the Neisseria gonorrhoeae pilus secretin protein PilQ allows increased entry of heme and antimicrobial compounds. J. Bacteriol. 186:730-739. [PMC free article] [PubMed]
5. Chen, I., and D. Dubnau. 2003. DNA transport during transformation. Front. Biosci. 8:s544-556. [PubMed]
6. Dietrich, G., S. Kurz, C. Hubner, C. Aepinus, S. Theiss, M. Guckenberger, U. Panzner, J. Weber, and M. Frosch. 2003. Transcriptome analysis of Neisseria meningitidis during infection. J. Bacteriol. 185:155-164. [PMC free article] [PubMed]
7. Du, Y., and C. G. Arvidson. 2006. RpoH mediates the expression of some, but not all, genes induced in Neisseria gonorrhoeae adherent to epithelial cells. Infect. Immun. 74:2767-2776. [PMC free article] [PubMed]
8. Du, Y., J. Lenz, and C. G. Arvidson. 2005. Global gene expression and the role of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells. Infect. Immun. 73:4834-4845. [PMC free article] [PubMed]
9. Edwards, J. L., and M. A. Apicella. 2004. The molecular mechanisms used by Neisseria gonorrhoeae to initiate infection differ between men and women. Clin. Microbiol. Rev. 17:965-981. [PMC free article] [PubMed]
10. Forest, K. T., K. A. Satyshur, G. A. Worzalla, J. K. Hansen, and T. J. Herdendorf. 2004. The pilus-retraction protein PilT: ultrastructure of the biological assembly. Acta Crystallogr. D 60:978-982. [PubMed]
11. Howie, H. L., M. Glogauer, and M. So. 2005. The N. gonorrhoeae type IV pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-dependent mechanism. PLOS Biol. 3:e100. [PMC free article] [PubMed]
12. Klose, K. E., and J. J. Mekalanos. 1997. Simultaneous prevention of glutamine synthesis and high-affinity transport attenuates Salmonella typhimurium virulence. Infect. Immun. 65:587-596. [PMC free article] [PubMed]
13. Lee, E. H., C. Rouquette-Loughlin, J. P. Folster, and W. M. Shafer. 2003. FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae via an MtrR regulatory mechanism. J. Bacteriol. 185:7145-7152. [PMC free article] [PubMed]
14. Lee, E. H., and W. M. Shafer. 1999. The farAB-encoded efflux pump mediates resistance of gonococci to long-chained antibacterial fatty acids. Mol. Microbiol. 33:839-845. [PubMed]
15. Lee, S. W., D. L. Higashi, A. Snyder, A. J. Merz, L. Potter, and M. So. 2005. PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. Cell Microbiol. 7:1271-1284. [PubMed]
16. Maier, B., L. Potter, M. So, C. D. Long, H. S. Seifert, and M. P. Sheetz. 2002. Single pilus motor forces exceed 100 pN. Proc. Natl. Acad. Sci. USA 99:16012-16017. [PMC free article] [PubMed]
17. Mattick, J. S. 2002. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56:289-314. [PubMed]
18. Merz, A. J., C. A. Enns, and M. So. 1999. Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells. Mol. Microbiol. 32:1316-1332. [PubMed]
19. Merz, A. J., and K. T. Forest. 2002. Bacterial surface motility: slime trails, grappling hooks and nozzles. Curr. Biol. 12:R297-303. [PubMed]
20. Merz, A. J., and M. So. 1997. Attachment of piliated, Opa and Opc gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins. Infect. Immun. 65:4341-4349. [PMC free article] [PubMed]
21. Merz, A. J., and M. So. 2000. Interactions of pathogenic neisseriae with epithelial cell membranes. Annu. Rev. Cell Dev. Biol. 16:423-457. [PubMed]
22. Merz, A. J., M. So, and M. P. Sheetz. 2000. Pilus retraction powers bacterial twitching motility. Nature 407:98-102. [PubMed]
23. Morand, P. C., E. Bille, S. Morelle, E. Eugene, J. L. Beretti, M. Wolfgang, T. F. Meyer, M. Koomey, and X. Nassif. 2004. Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins. EMBO J. 23:2009-2017. [PMC free article] [PubMed]
24. Nassif, X., C. Pujol, P. Morand, and E. Eugene. 1999. Interactions of pathogenic Neisseria with host cells. Is it possible to assemble the puzzle? Mol. Microbiol. 32:1124-1132. [PubMed]
25. Nohno, T., T. Saito, and J. S. Hong. 1986. Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ). Mol. Gen. Genet. 205:260-269. [PubMed]
26. Oh, J. D., H. Kling-Backhed, M. Giannakis, J. Xu, R. S. Fulton, L. A. Fulton, H. S. Cordum, C. Wang, G. Elliott, J. Edwards, E. R. Mardis, L. G. Engstrand, and J. I. Gordon. 2006. The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression. Proc. Natl. Acad. Sci. USA 103:9999-10004. [PMC free article] [PubMed]
27. Okamoto, S., and M. Ohmori. 2002. The cyanobacterial PilT protein responsible for cell motility and transformation hydrolyzes ATP. Plant Cell Physiol. 43:1127-1136. [PubMed]
28. Schleif, R. 1996. Two positively regulated systems, ara and mal, p. 1300-1309. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, DC.
29. Sexton, J. A., J. S. Pinkner, R. Roth, J. E. Heuser, S. J. Hultgren, and J. P. Vogel. 2004. The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth. J. Bacteriol. 186:1658-1666. [PMC free article] [PubMed]
30. Stabler, R. A., G. L. Marsden, A. A. Witney, Y. Li, S. D. Bentley, C. M. Tang, and J. Hinds. 2005. Identification of pathogen-specific genes through microarray analysis of pathogenic and commensal Neisseria species. Microbiology 151:2907-2922. [PubMed]
31. Tamura, G. S., A. Nittayajarn, and D. L. Schoentag. 2002. A glutamine transport gene, glnQ, is required for fibronectin adherence and virulence of group B streptococci. Infect. Immun. 70:2877-2885. [PMC free article] [PubMed]
32. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547. [PubMed]
33. Vale, R. D. 2000. AAA proteins. Lords of the ring. J. Cell Biol. 150:F13-F19. [PMC free article] [PubMed]
34. Varga, J. J., V. Nguyen, D. K. O'Brien, K. Rodgers, R. A. Walker, and S. B. Melville. 2006. Type IV pili-dependent gliding motility in the Gram-positive pathogen Clostridium perfringens and other clostridia. Mol. Microbiol. 62:680-694. [PubMed]
35. Veal, W. L., and W. M. Shafer. 2003. Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae. J. Antimicrob. Chemother. 51:27-37. [PubMed]
36. Wall, D., and D. Kaiser. 1999. Type IV pili and cell motility. Mol. Microbiol. 32:1-10. [PubMed]
37. Wolfgang, M., P. Lauer, H. S. Park, L. Brossay, J. Hebert, and M. Koomey. 1998. PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol. Microbiol. 29:321-330. [PubMed]

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