Cindy Grove Arvidson

Lactobacillus jensenii surface-associated proteins inhibit Neisseria gonorrhoeae adherence to epithelial cells

ABSTRACT High numbers of lactobacilli in the vaginal tract have been correlated with a decreased risk of infection by the sexually transmitted pathogen Neisseria gonorrhoeae. We have previously shown that Lactobacillus jensenii, one of the most prevalent microorganisms in the healthy human vaginal tract, can inhibit gonococcal adherence to epithelial cells in culture. Here we examined the role of the epithelial cells and the components of L. jensenii involved in the inhibition of gonococcal adherence. L. jensenii inhibited the adherence of gonococci to glutaraldehyde-fixed epithelial cells like it inhibited the adherence of gonococci to live epithelial cells, suggesting that the epithelial cells do not need to be metabolically active for the inhibition to occur. In addition, methanol-fixed L. jensenii inhibited gonococcal adherence to live epithelial cells, indicating that L. jensenii uses a constitutive component to inhibit gonococcal interactions with epithelial cells. Proteinase K treatment of methanol-fixed lactobacilli eliminated the inhibitory effect, suggesting that the inhibitory component contains protein. Released surface components (RSC) isolated from L. jensenii were found to contain at least two inhibitory components, both of which are protease sensitive. Using anion-exchange and size exclusion chromatography, an inhibitory protein which exhibits significant similarity to the enzyme enolase was isolated. A recombinant His6-tagged version of this protein was subsequently produced and shown to inhibit gonococcal adherence to epithelial cells in a dose-dependent manner.

Inhibition of Neisseria gonorrhoeae epithelial cell interactions by vaginal Lactobacillus species.

High levels of Lactobacillus, the dominant genus of the healthy human vaginal microbiota, have been epidemiologically linked to a reduced risk of infection following exposure to the sexually transmitted pathogen Neisseria gonorrhoeae. In this work, a cell culture model of gonococcal infection was adapted to examine the effects of lactobacilli on gonococcal interactions with endometrial epithelial cells in vitro. Precolonization of epithelial cells with Lactobacillus jensenii, Lactobacillus gasseri ATCC 33323, or L. gasseri ATCC 9857 reduced gonococcal adherence by nearly 50%. Lactobacilli also inhibited gonococcal invasion of epithelial cells by more than 60%, which was independent of the effect on adherence. Furthermore, lactobacilli were able to displace adherent gonococci from epithelial cells, suggesting that these organisms have potential as a postexposure prophylactic. Thus, vaginal lactobacilli have the ability to inhibit gonococci at two key steps of an infection, which might have a significant effect in determining whether the gonococcus will be able to successfully establish an infection following exposure in vivo.

Lactobacilli at the front line of defense against vaginally acquired infections

Probiotics are microorganisms that provide a health benefit to the host and are promoted as alternatives for the treatment and prevention of infectious diseases and other conditions. One of the most rapidly developing areas of probiotic research is in the management of vaginally acquired infections. Several Lactobacillus species produce compounds that kill or inhibit the growth of vaginally acquired pathogens. Other lactobacilli reduce the adherence of pathogens to urogenital epithelial cells in culture. This article discusses the mechanisms by which vaginal lactobacilli prevent pathogen colonization of the urogenital tract, and potential mechanisms that warrant investigation. Animal models and clinical studies, while limited, are discussed with the idea that these are the next critical steps to advance the study of probiotics for the treatment and prevention of vaginally acquired infections.

Global Gene Expression and the Role of Sigma Factors in Neisseria gonorrhoeae in Interactions with Epithelial Cells

ABSTRACT Like many bacterial pathogens, Neisseria gonorrhoeae must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in N. gonorrhoeae in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was rpoH, which encodes a homolog of the heat shock sigma factor, σ32 (RpoH), as well as genes of the RpoH regulon, groEL and groES. Attempts to construct an rpoH null mutant in N. gonorrhoeae were unsuccessful, suggesting that RpoH is essential for viability of N. gonorrhoeae. The extracytoplasmic sigma factor, RpoE (σE), while known to regulate rpoH in other bacteria, was found not to be necessary for the up-regulation of rpoH in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an N. gonorrhoeae strain conditionally expressing rpoH was constructed. The results of our experiments showed that while induction of rpoH expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for rpoH invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that σ32, but not σE, is important for the response of gonococci in the initial steps of an infection.

Attenuated enzootic (pestoides) isolates of Yersinia pestis express active aspartase

It is established that Yersinia pestis, the causative agent of bubonic plague, recently evolved from enteropathogenic Yersinia pseudotuberculosis by undergoing chromosomal degeneration while acquiring two unique plasmids that facilitate tissue invasion (pPCP) and dissemination by fleabite (pMT). Thereafter, plague bacilli spread from central Asia to sylvatic foci throughout the world. These epidemic isolates exhibit a broad host range including man as opposed to enzootic (pestoides) variants that remain in ancient reservoirs where infection is limited to muroid rodents. Cells of Y. pseudotuberculosis are known to express glucose-6-phosphate dehydrogenase (Zwf) and aspartase (AspA); these activities are not detectable in epidemic Y. pestis due to missense mutations (substitution of proline for serine at amino position 155 of Zwf and leucine for valine at position 363 of AspA). In this study, functional Zwf was found in pestoides strains E, F and G but not seven other enzootic isolates; enzymic activity was associated with retention of serine at amino acid position 155. Essentially, full AspA activity occurred in pestoides isolates where valine (pestoides A, B, C and D) or serine (pestoides E, F, G and I) occupied position 363. Reduced activity occurred in strains Angola and A16, which contained phenylalanine at this position. The kcat but not Km of purified AspA from strain Angola was significantly reduced. In this context, aspA of the recently described attenuated enzootic microtus biovar encodes active valine at position 363, further indicating that functional AspA is a biomarker for avirulence of Y. pestis in man.

Increased Expression of the Type IV Secretion System in Piliated Neisseria gonorrhoeae Variants

Neisseria gonorrhoeae produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either traD expression or growth rate. A mutant with a deletion in traD was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that traD is transcriptionally and translationally coupled to traI, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing traI and traD. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of traD and traI.

Expression of a Clostridium perfringens Type IV Pilin by Neisseria gonorrhoeae Mediates Adherence to Muscle Cells

ABSTRACT Clostridium perfringens is an anaerobic, Gram-positive bacterium that causes a range of diseases in humans, including lethal gas gangrene. We have recently shown that strains of C. perfringens move across the surface of agar plates by a unique type IV pilus (TFP)-mediated social motility that had not been previously described. Based on sequence homology to pilins in Gram-negative bacteria, C. perfringens appears to have two pilin subunits, PilA1 and PilA2. Structural prediction analysis indicated PilA1 is similar to the pseudopilin found in Klebsiella oxytoca, while PilA2 is more similar to true pilins found in the Gram-negative pathogens Pseudomonas aeruginosa and Neisseria gonorrhoeae. Strains of N. gonorrhoeae that were genetically deficient in the native pilin, PilE, but supplemented with inducible expression of PilA1 and PilA2 of C. perfringens were constructed. Genetic competence, wild-type twitching motility, and attachment to human urogenital epithelial cells were not restored by expression of either pilin. However, attachment to mouse and rat myoblast (muscle) cell lines was observed with the N. gonorrhoeae strain expressing PilA2. Significantly, wild-type C. perfringens cells adhered to mouse myoblasts under anaerobic conditions, and adherence was 10-fold lower in a pilT mutant that lacked functional TFP. These findings implicate C. perfringens TFP in the ability of C. perfringens to adhere to and move along muscle fibers in vivo, which may provide a therapeutic approach to limiting this rapidly spreading and highly lethal infection.

A missense mutation causes aspartase deficiency in Yersinia pestis

It is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete l-aspartic acid at the expense of exogenous l-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G.C-->T.A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G.C-->T.A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coli. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for l-aspartic acid. However, the k(cat) of the Y. pestis CRIM of strain KIM is only about 0.1 % of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover (k(cat) 86+/-2 s(-1)) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.

Two ABC Transporter Operons and the Antimicrobial Resistance Gene mtrF Are pilT Responsive in Neisseria gonorrhoeae

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.

The Neisseria transcriptional regulator PilA has a GTPase activity

The pilA gene of Neisseria gonorrhoeae encodes the response regulator of a two-component regulatory system that controls pilin gene expression. Examination of the primary sequence of PilA indicates that the protein contains at least two functional domains. The N-terminal region has a proposed helix-turn-helix motif thought to be involved in DNA binding. This region also contains the residues that are presumed to form the acidic pocket involved in phosphorylation by PilB, the sensor kinase of the system. The C terminus of the protein has extensive homology to the G (GTP-binding) domains of the eukaryotic signal recognition particle (SRP) 54-kDa protein and the α subunit of the SRP receptor, or docking protein. This homology also extends to similar regions of the bacterial SRP homologs Ffh and FtsY. Here, we demonstrate that purified PilA has significant GTPase activity, and that this activity has an absolute requirement for MgCl2 and is sensitive to KCl and low pH. We also show that PilA has a strict specificity for GTP, and that GTP hydrolysis follows first order kinetics, with a maximum velocity (Vmax) of 1900 pmol of P produced per min per mg of protein and a K for GTP of 9.6 μM at 37°C.