The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three \'noNterm\' Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

The effects of intestinal microflora on extraintestinal immune response by intestinal cytokines and metabolites have been documented, but whether intestinal microbes stimulate serum antibody generation is unknown. Here, serum antibodies against 69 outer membrane proteins of Escherichia coli, a dominant bacterium in the human intestine, are detected in 141 healthy individuals of varying ages. Antibodies against E. coli outer membrane proteins are determined in all serum samples tested, and frequencies of antibodies to five outer membrane proteins (OmpA, OmpX, TsX, HlpA, and FepA) are close to 100%. Serum antibodies against E. coli outer membrane proteins are further validated by Western blot and bacterial pull-down. Moreover, the present study shows that OstA, HlpA, Tsx, NlpB, OmpC, YfcU, and OmpA provide specific immune protection against pathogenic E. coli, while HlpA and OmpA also exhibit cross-protection against Staphylococcus aureus infection. These finding indicate that intestinal E. coli activate extraintestinal antibody responses and provide anti-infective immunity.

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 β-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.
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.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

The biogenesis of outer membrane proteins is mediated by the β-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in Escherichia coli. Despite great progress in the BAM structural analysis, the molecular details of BAM-mediated processes as well as the exact function of each BAM component during OMP assembly are still not fully understood. To enable a distinguishment of the function of each BAM component, it is the aim of the present work to examine and identify the effective minimum form of the E. coli BAM complex by use of a well-defined reconstitution strategy based on a previously developed versatile assay. Our data demonstrate that BamADE is the core BAM component and constitutes a minimum functional form for OMP assembly in E. coli, which can be stimulated by BamB and BamC. While BamB and BamC have a redundant function based on the minimum form, both together seem to cooperate with each other to substitute for the function of the missing BamD or BamE. Moreover, the BamAE470K mutant also requires the function of BamD and BamE to assemble OMPs in vitro, which vice verse suggests that BamADE are the effective minimum functional form of the E. coli BAM complex.

Treponema pallidum, the causative agent of syphilis, is a sexually transmitted microorganism that exhibits remarkable motility capabilities, allowing it to affect various systems. Despite its structural resemblance to gram-negative bacteria due to its dual-membrane, T. pallidum possesses a lower abundance of outer membrane proteins (OMPs), which enables it to effectively conceal itself. This review presents a comprehensive analysis of the clinical diagnostic potential associated with the OMPs of T. pallidum. Furthermore, the known OMPs in T. pallidum that are responsible for mediating host interactions have been progressively elucidated. This review aims to shed light on the pathogenesis of syphilis, encompassing aspects such as vascular inflammation, chancre self-healing, neuroinvasion, and reinfection. Additionally, this review offers a detailed overview of the current state and prospects of development in the field of syphilis vaccines, with the ultimate goal of establishing a foundation for understanding the pathogenesis and implementing effective prevention strategies against syphilis.

Previous mass spectrometry (MS)-based global proteomics studies have detected a combined total of 86% of all Treponema pallidum proteins under infection conditions (in vivo-grown T. pallidum). Recently, a method was developed for the long-term culture of T. pallidum under in vitro conditions (in vitro-cultured T. pallidum). Herein, we used our previously reported optimized MS-based proteomics approach to characterize the T. pallidum global protein expression profile under in vitro culture conditions. These analyses provided a proteome coverage of 94%, which extends the combined T. pallidum proteome coverage from the previously reported 86% to a new combined total of 95%. This study provides a more complete understanding of the protein repertoire of T. pallidum. Further, comparison of the in vitro-expressed proteome with the previously determined in vivo-expressed proteome identifies only a few proteomic changes between the two growth conditions, reinforcing the suitability of in vitro-cultured T. pallidum as an alternative to rabbit-based treponemal growth. The MS proteomics data have been deposited in the MassIVE repository with the data set identifier MSV000093603 (ProteomeXchange identifier PXD047625).

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the β-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand β-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling β-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of β-barrel protein modeling: how different types of molecules can diffuse through β-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how β-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).