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Associations with symbionts within the gut lumen of hosts are particularly prone to disruption due to the constant influx of ingested food and non-symbiotic microbes, yet we know little about how partner fidelity is maintained. Here we describe for the first time the existence of a gut morphological filter capable of protecting an animal gut microbiome from disruption. The proventriculus, a valve located between the crop and midgut of insects, functions as a micro-pore filter in the Sonoran Desert turtle ant ( Cephalotes rohweri ), blocking the entry of bacteria and particles ⩾0.2 μm into the midgut and hindgut while allowing passage of dissolved nutrients. Initial establishment of symbiotic gut bacteria occurs within the first few hours after pupation via oral–rectal trophallaxis, before the proventricular filter develops. Cephalotes ants are remarkable for having maintained a consistent core gut microbiome over evolutionary time and this partner fidelity is likely enabled by the proventricular filtering mechanism. In addition, the structure and function of the cephalotine proventriculus offers a new perspective on organismal resistance to pathogenic microbes, structuring of gut microbial communities, and development and maintenance of host–microbe fidelity both during the animal life cycle and over evolutionary time.

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.

Retraction of the pathogenic Neisseria Type IV pilus (Tfp) requires PilT, the primary retraction motor, and its paralogs PilU and PilT2. The importance of Tfp retraction for natural infection is unknown. Using our natural animal model of Neisseria -host interaction, we have examined the role of these proteins in the ability of commensal Neisseria musculi (Nmus) to colonize and persist in its native host, the mouse. We report that Nmus Δ pilT cannot colonize mice; Δ pilU and Δ pilT2 can colonize and persist in mice, but in lower numbers. Microcolonies formed by Δ pilT , Δ pilTU , and pilT L201C , expressing PilT with a point mutation in its ATP hydrolysis domain, are sensitive to removal by fluid shear forces. Thus, PilT promotes Nmus colonization while PilU and PilT2 influence persistence. We present a non-exclusive model for how these Tfp retraction motor proteins contribute to Nmus persistent colonization. Our findings have implications for the roles of these motor proteins in mediating interactions of human-adapted pathogenic and commensal Neisseria with their human host. We describe the importance of Type IV pilus retraction to colonization and persistence by a mouse commensal Neisseria, N. musculi, in its native host. Our findings have implications for the role of Tfp retraction in mediating interactions of human-adapted pathogenic and commensal Neisseria with their human host due to the relatedness of these species.

Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. Δ pilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilT L201C ). Purified PilT L201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilT L201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilT L201C cells is intermediate between the behaviors of wt and Δ pilT cells. The infection behavior of pilT L201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se , is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology. IMPORTANCE Type IV pili are fibers expressed on the surface of many bacteria. Neisseria gonorrhoeae cells crawl, take up DNA, and communicate with each other and with human cells by retracting these fibers. Here, we show that an N. gonorrhoeae mutant expressing an enzymatically weakened type IV pilus retraction motor still crawls and takes up DNA normally. However, mutant cells exhibit abnormal social behavior, and they are less infective because they fail to activate the epidermal growth factor receptor. Our study shows that N. gonorrhoeae social and infection behaviors are sensitive to the strength of the retraction motor enzyme. Type IV pili are fibers expressed on the surface of many bacteria. Neisseria gonorrhoeae cells crawl, take up DNA, and communicate with each other and with human cells by retracting these fibers. Here, we show that an N. gonorrhoeae mutant expressing an enzymatically weakened type IV pilus retraction motor still crawls and takes up DNA normally. However, mutant cells exhibit abnormal social behavior, and they are less infective because they fail to activate the epidermal growth factor receptor. Our study shows that N. gonorrhoeae social and infection behaviors are sensitive to the strength of the retraction motor enzyme.

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species.

We identified and characterized 14 novel short-tandem-repeats (STRs) on the Y chromosome and typed them in two samples, a globally diverse panel of 73 cell lines, and 148 individuals from a European–American population. These Y-STRs include eight tetranucleotide repeats (DYS449, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459, and DYS464), five pentanucleotide repeats (DYS446, DYS447, DYS450, DYS452, and DYS463), and one hexanucleotide repeat (DYS448). Sequence data were obtained to designate a repeat number nomenclature. The gene diversities of an additional 22 Y-STRs, including the most commonly used in forensic databases, were directly compared in the cell line DNAs. Six of the 10 most polymorphic markers include the newly identified Y-STRs. Furthermore, these novel Y-STRs greatly improved the resolution of paternal lineages, above the level obtained with commonly used Y-STRs, in the European–American population.