Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae . Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω ^{tox +} phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox ⁺ corynephage. DNA binding sites of the tox -controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

Clostridium difficile is a Gram-positive, spore-forming anaerobic human gastrointestinal pathogen. C. difficile infection (CDI) is a major health concern worldwide, with symptoms ranging from diarrhea to pseudomembranous colitis, toxic megacolon, sepsis, and death. CDI onset and progression are mostly caused by intestinal dysbiosis and exposure to C. difficile spores. Current treatment strategies include antibiotics; however, antibiotic use is often associated with high recurrence rates and an increased risk of antibiotic resistance. Medium-chain fatty acids (MCFAs) have been revealed to inhibit the growth of multiple human bacterial pathogens. Components of coconut oil, which include lauric acid, have been revealed to inhibit C. difficile growth in vitro. In this study, we demonstrated that lauric acid exhibits potent antimicrobial activities against multiple toxigenic C. difficile isolates in vitro. The inhibitory effect of lauric acid is partly due to reactive oxygen species (ROS) generation and cell membrane damage. The administration of lauric acid considerably reduced biofilm formation and preformed biofilms in a dose-dependent manner. Importantly, in a mouse infection model, lauric acid pretreatment reduced CDI symptoms and proinflammatory cytokine production. Our combined results suggest that the naturally occurring MCFA lauric acid is a novel C. difficile inhibitor and is useful in the development of an alternative or adjunctive treatment for CDI.

The oral biofilm is a multispecies community in which antagonism and mutualism coexist among friends and foes to keep an ecological balance of community members. The pioneer colonizers, such asStreptococcus gordonii, produce H₂O₂to inhibit the growth of competitors, like the mutans streptococci, as well as strict anaerobic middle and later colonizers of the dental biofilm. Interestingly,Veillonellaspecies, as early colonizers, physically interact (coaggregate) withS. gordonii. A putative catalase gene (catA) is found in most sequencedVeillonellaspecies; however, the function of this gene is unknown. In this study, we characterized the ecological function ofcatAfromVeillonella parvulaPK1910 by integrating it into the only transformable strain,Veillonella atypicaOK5, which iscatAnegative. The strain (OK5-catA) became more resistant to H₂O₂. Further studies demonstrated that thecatAgene expression is induced by the addition of H₂O₂or coculture withS. gordonii. Mixed-culture experiments further revealed that the transgenic OK5-catAstrain not only enhanced the growth ofFusobacterium nucleatum, a strict anaerobic periodontopathogen, under microaerophilic conditions, but it also rescuedF. nucleatumfrom killing byS. gordonii. A potential role of catalase in veillonellae in biofilm ecology and pathogenesis is discussed here.IMPORTANCEVeillonellaspecies, as early colonizers, can coaggregate with many bacteria, including the initial colonizerStreptococcus gordoniiand periodontal pathogenFusobacterium nucleatum, during various stages of oral biofilm formation. In addition to providing binding sites for many microbes, our previous study also showed thatVeillonellaproduces nutrients for the survival and growth of periodontal pathogens. These findings indicate thatVeillonellaplays an important “bridging” role in the development of oral biofilms and the ecology of the human oral cavity. In this study, we demonstrated that the reducing activity ofVeillonellacan rescue the growth ofFusobacterium nucleatumnot only under microaerophilic conditions, but also in an environment in whichStreptococcus gordoniiis present. Thus, this study will provide a new insight for future studies on the mechanisms of human oral biofilm formation and the control of periodontal diseases.

In the oral biofilm, the ‘mitis’ streptococci are among the first group of organisms to colonize the tooth surface. Their proliferation is thought to be an important factor required for antagonizing the growth of cariogenic species such asStreptococcus mutans. In this study, we used a three-species mixed culture to demonstrate that another ubiquitous early colonizing species,Veillonella parvula,can greatly affect the outcome of the competition between a pair of antagonists such asS. mutansandStreptococcus gordonii. Transcriptome analysis further revealed thatS. mutansresponds differentially to its friend (V. parvula) and foe (S. gordonii). In the mixed culture withS. gordonii, all but one of theS. mutanssugar uptake and metabolic genes were downregulated, while genes for alternative energy source utilization and H₂O₂tolerance were upregulated, resulting in a slower but persistent growth. In contrast, when cultured withV. parvula,S. mutansgrew equally well or better than in monoculture and exhibited relatively few changes within its transcriptome. WhenV. parvulawas introduced into the mixed culture ofS. mutansandS. gordonii, it rescued the growth inhibition ofS. mutans. In this three-species environment,S. mutansincreased the expression of genes required for the uptake and metabolism of minor sugars, while genes required for oxidative stress tolerance were downregulated. We conclude that the major factors that affect the competition betweenS. mutansandS. gordoniiare carbohydrate utilization and H₂O₂resistance. The presence ofV. parvulain the tri-species culture mitigates these two major factors and allowsS. mutansto proliferate, despite the presence ofS. gordonii.

Summary: Sortase, a cysteine‐transpeptidase conserved in Gram‐positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram‐positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall‐anchored protein substrates (AcaA‐N) were individually dispensable for cell viability. Using Tn5‐transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR‐CpsA‐Psr (LCP)‐like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane‐localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco‐stress provides convenient cell‐based assays for developing a new class of inhibitors against Gram‐positive pathogens.

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced C. perfringens strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on C. perfringens gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human- and animal-derived pathogenic C. perfringens strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in C. perfringens , such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes pilT and pilD , whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on pilT and pilD expression were under the control of the key regulatory protein CcpA ( c atabolite c ontrol p rotein A). The deficiency in CcpA activity of a ccpA knockout C. perfringens mutant strain restored the expressions of pilT and pilD and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the ccpA mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of ccpA . These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on C. perfringens gliding motility. Furthermore, we discovered a novel positive role for CcpA in pilT and pilD expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by C. perfringens .