Prevalence of Slam-dependent hemophilins in Gram-negative bacteria.

Iron acquisition systems are crucial for pathogen growth and survival in iron-limiting host environments. To overcome nutritional immunity, bacterial pathogens evolved to use diverse mechanisms to acquire iron. Here, we examine a heme acquisition system that utilizes hemophores called hemophilins which are also referred to as HphAs in several Gram-negative bacteria. In this study, we report three new HphA structures from Stenotrophomonas maltophilia, Vibrio harveyi, and Haemophilus parainfluenzae. Structural determination of HphAs revealed an N-terminal clamp-like domain that binds heme and a C-terminal eight-stranded ß-barrel domain that shares the same architecture as the Slam-dependent Neisserial surface lipoproteins. The genetic organization of HphAs consists of genes encoding a Slam homolog and a TonB-dependent receptor (TBDR). We investigated the Slam-HphA system in the native organism or the reconstituted system in Escherichia coli cells and found that the efficient secretion of HphA depends on Slam. The TBDR also played an important role in heme uptake and conferred specificity for its cognate HphA. Furthermore, bioinformatic analysis of HphA homologs revealed that HphAs are conserved in the alpha, beta, and gammaproteobacteria. Together, these results show that the Slam-dependent HphA-type hemophores are prevalent in Gram-negative bacteria and further expand the role of Slams in transporting soluble proteins. IMPORTANCE: This paper describes the structure and function of a family of Slam (Type IX secretion System) secreted hemophores that bacteria use to uptake heme (iron) while establishing an infection. Using structure-based bioinformatics analysis to define the diversity and prevalence of this heme acquisition pathway, we discovered that a large portion of gammaproteobacterial harbors this system. As organisms, including Acinetobacter baumannii, utilize this system to facilitate survival during host invasion, the identification of this heme acquisition system in bacteria species is valuable information and may represent a target for antimicrobials.

Gut commensal Enterocloster species host inoviruses that are secreted in vitro and in vivo.

BACKGROUND: Bacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized. RESULTS: In this study, we utilized in silico, in vitro, and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. (formerly Clostridium spp.). We confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated bacterial growth dynamics, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives. CONCLUSION: In this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria. Video Abstract.