Natural transformation is a dominant force in bacterial evolution by promoting horizontal gene transfer. This process may have devastating consequences, such as the spread of antibiotic resistance or the emergence of highly virulent clones. However, uptake and recombination of foreign DNA are most often deleterious to competent species. Therefore, model naturally transformable Gramnegative bacteria, including the human pathogen Neisseria meningitidis, have evolved means to preferentially take up homotypic DNA containing short and genus-specific sequence motifs. Despite decades of intense investigations, the DNA uptake sequence receptor in Neisseria species has remained elusive. We show here, using a multidisciplinary approach combining biochemistry, molecular genetics, and structural biology, that meningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filaments surface and that ComP displays an exquisite binding preference for DNA uptake sequence. Our findings illuminate the earliest step in natural transformation, reveal an unconventional mechanism for DNA binding, and suggest that selective DNA uptake is more widespread than previously thought.DNA receptor | genetic competence

Background Gonorrhea, caused by the bacterium Neisseria gonorrhoeae, is a globally prevalent sexually transmitted infection. The dynamics of gonococcal population biology have been poorly defined due to a lack of resolution in strain typing methods. Methods In this study, we assess how the core genome can be used to improve our understanding of gonococcal population structure compared with current typing schemes. Results A total of 1668 loci were identified as core to the gonococcal genome. These were organized into a core genome multilocus sequence typing scheme (N gonorrhoeae cgMLST v1.0). A clustering algorithm using a threshold of 400 allelic differences between isolates resolved gonococci into discrete and stable core genome groups, some of which persisted for multiple decades. These groups were associated with antimicrobial genotypes and non-overlapping NG-STAR and NG-MAST sequence types. The MLST-STs were more widely distributed among core genome groups. Conclusions Clustering with cgMLST identified globally distributed, persistent, gonococcal lineages improving understanding of the population biology of gonococci and revealing its population structure. These findings have implications for the emergence of antimicrobial resistance in gonococci and how this is associated with lineages, some of which are more predisposed to developing antimicrobial resistance than others.

Natural transformation is the widespread biological process by which “competent” bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or “transformed”. To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence) watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.

Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophagelike cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophagelike cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4 ؊/؊ mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.Mycobacterium tuberculosis remains a major global pathogen killing an estimated 2 million people each year (32). Young and coworkers have recently stressed the urgent need to understand how this organism causes disease and to identify the key virulence factors (35). One of the earliest described factors from M. tuberculosis, and one potentially involved in the pathogenesis of tuberculosis, was the heat shock protein chaperonin 60 (Cpn60) or heat shock protein 60 (Hsp60). This was initially identified as a "common antigen" to which antibodies were raised in a variety of bacterial infections and later identified as the molecular chaperone homologous to Escherichia coli GroEL and termed Hsp65 (15). The Hsp65 protein of the mycobacteria has been the subject of enormous interest because of its immunomodulatory influence on T-cell immunity (31). These effects were thought to be due to Hsp65 acting as an immunogen. However, Friedland et al. revealed that M. tuberculosis Hsp65 was able to directly activate human monocyte proinflammatory cytokine production (5) and generated the hypothesis, which is still the subject of some controversy, that chaperonin 60 proteins are moonlighting proteins (11) that can be secreted from cells and have cell-cell signaling functions (1, 18).M. tuberculosis is one of the growing propor...

BackgroundAfrica has the highest incidence of gonorrhea in the world. However, little is known about gonococcal populations in this continent or mechanisms of antimicrobial resistance (AMR).MethodsWhole-genome sequence data were analyzed from 103 Neisseria gonorrhoeae isolates from 73 patients, mainly men who have sex with men, from coastal Kenya. We annotated loci, defined the core genome, defined mechanisms of AMR, and performed phylogenetic analysis. For patients with multiple episodes of gonorrhea, we determined whether infections occurred with related strains.ResultsWe identified 3 clusters of isolates that are phylogenetically distinct from isolates found elsewhere. Plasmids were virtually ubiquitous: pTetM and pblaTEM were found in 97%, and 55% of isolates, respectively. This was associated with high doxycycline use for undiagnosed sexually transmitted infections. Twenty-three percent of multiple episodes of gonorrhea in the same individual were caused by a related strain, suggesting inadequate treatment or reinfection.ConclusionsThe prevalence of plasmid-mediated AMR in Kenyan gonococci contrasts with that in wealthy countries, where AMR is largely chromosomally mediated. Antimicrobials have a profound effect on the maintenance of lineages harboring plasmids. Doxycycline can select for tetracycline and penicillin resistance, through plasmid cooperation. Understanding the mechanisms of AMR in high-risk groups is required to inform treatment strategies.

Plasmids are vehicles for horizontal gene transfer between bacteria, and in Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance (AMR). Using genomic and phylogenetic analyses, we show that plasmids are widespread in a collection of 3724 gonococcal isolates from 56 countries, and characterized the conjugative, β-lactamase and cryptic plasmids. We found that variants of the conjugative plasmid (which can mediate tetracycline resistance) and the β-lactamase plasmid expressing TEM-135 are associated with distinct gonococcal lineages. Furthermore, AMR plasmids are significantly more prevalent in gonococci from less wealthy countries, highlighting the need for further studies. More than 94% of gonococci possess the cryptic plasmid, with its absence correlated with the presence of a novel chromosomal type IV secretion system. Our results reveal the extent of plasmid-mediated AMR in the gonococcus, particularly in less wealthy countries, where diagnostic and therapeutic options can be limited, and highlight the risk of their global spread.

The rapid onset and dramatic consequences of Neisseria meningitidis infections make the design of a broadly protective vaccine a priority for public health. There is an ongoing quest for meningococcal components that are surface exposed, widely conserved and can induce protective antibodies. Type IV pili (Tfp) are filamentous structures with a key role in pathogenesis that extend beyond the surface of the bacteria and have demonstrated vaccine potential. However, extensive antigenic variation of PilE, the major subunit of Tfp, means that they are currently considered to be unsuitable vaccine components. Recently it has been shown that Tfp also contain low abundance pilins ComP, PilV and PilX in addition to PilE. This prompted us to examine the prevalence and sequence diversity of these proteins in a panel of N. meningitidis disease isolates. We found that all minor pilins are highly conserved and the major pilin genes are also highly conserved within the ST-8 and ST-11 clonal complexes. These data have important implications for the re-consideration of pilus subunits as vaccine antigens.

To test the hypothesis that prolonged culture would enhance the sensitivity of latent tuberculosis detection by a gamma interferon release assay, blood samples from 33 household contacts of Gambian tuberculosis patients were stimulated with Mycobacterium tuberculosis-specific antigens. After 24 h of culture, 66% were positive, compared to 93% after 6 days of culture.