TseVF-TsiVF, a novel bacteriolytic effector-immunity pair of Vibrio fluvialis VflT6SS2, provides a fitness advantage in microbial competition

Crystal structure analysis reveals a novel forkhead‐associated domain of ESAT‐6 secretion system C protein in <i>Staphylococcus aureus</i>

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.

Bacterial Nanotubes for Intimate Sharing

Vibrio fluvialis is an emerging foodborne pathogen causing gastroenteritis and extraintestinal infections, representing a significant public health concern due to rising antimicrobial resistance and the absence of an approved vaccine. This study aimed to design a multi-epitope subunit vaccine against V. fluvialis using immunoinformatics and a standard multi-epitope vaccine design pipeline. Two surface-exposed immunogenic membrane proteins, ATP-dependent zinc metalloprotease FtsH and lytic murein transglycosylase F, were selected as antigenic targets. Ten epitopes, including four MHC class I, four MHC class II, and two B-cell epitopes, were predicted and assembled into a 246 amino acid vaccine construct. The construct showed an antigenicity score of 0.8610. Population coverage analysis indicated that these epitopes could potentially cover 99.97% of the global population. The vaccine exhibited favorable physicochemical properties, with an instability index of 33.18 and a GRAVY score of - 0.282, suggesting stability and hydrophilicity. The tertiary structure was modeled using AlphaFold3 and docked with Toll-like receptor 2, yielding a docking score of - 270.01. Molecular dynamics simulations for 100 ns suggested stability of the vaccine-TLR2 complex. Codon optimization indicated high expression potential in Escherichia coli, with a CAI value of 0.95. Overall, the vaccine showed strong in silico immunogenic potential and requires further experimental validation through in vitro and in vivo studies.

The VirB secretion apparatus in Brucella belongs to the type IV secretion systems present in many pathogenic bacteria and is absolutely necessary for the efficient evasion of the Brucella-containing vacuole from the phagocytic route in professional phagocytes. This system is responsible for the secretion of a plethora of effector proteins that alter the biology of the host cell and promote the intracellular replication process. Although many VirB substrates have been identified in Brucella, we still know very little about the secretion mechanism that mediates their translocation across the two membranes and the periplasmic space. In this manuscript, we describe the identification of a gene, virJ, that codes for a protein with periplasmic localization that is involved in the intracellular replication process and virulence in mice. Our analysis revealed that this protein is necessary for the secretion of at least two VirB substrates that have a periplasmic intermediate and that it directly interacts with them. We additionally show that VirJ also associates with the apparatus per se and that its absence affects the assembly of the complex. We hypothesize that VirJ is part of a secretion platform composed of the translocon and several secretion substrates and that it probably coordinates the proper assembly of this macromolecular complex.

A Soluble Form of the Pilus Protein FimA Targets the VDAC-Hexokinase Complex at Mitochondria to Suppress Host Cell Apoptosis

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.

Type III secretion systems are central to the pathogenesis and virulence of many important Gram-negative bacterial pathogens, and elucidation of the secretion mechanism and identification of the secreted substrates are critical to our understanding of their pathogenic mechanisms and developing potential therapeutics. Stable isotope labeling with amino acids in cell culture-based mass spectrometry is a quantitative and highly sensitive proteomics tool that we have previously used to successfully analyze the type III secretomes of Citrobacter rodentium and Salmonella enterica serovar Typhimurium. In this report, stable isotope labeling with amino acids in cell culture was used to analyze the type III secretome of enteropathogenic Escherichia coli (EPEC), an important human pathogen, which, together with enterohemorrhagic E. coli and C. rodentium, represents the family of attaching and effacing bacterial pathogens. We not only confirmed all 25 known EPEC type III-secreted proteins and effectors previously identified by conventional molecular and bioinformatical techniques but also identified several new type III-secreted proteins, including two novel effectors, C_0814/NleJ and LifA, that were shown to be translocated into host cells. LifA is a known virulence factor believed to act as a toxin as well as an adhesin, but its mechanism of secretion and function is not understood. With a predicted molecular mass of 366 kDa, LifA is the largest type III effector identified thus far in any pathogen. We further demonstrated that Efa1, ToxB, and Z4332 (homologs of LifA in enterohemorrhagic E. coli) are also type III effectors. This study has comprehensively characterized the type III secretome of EPEC, expanded the repertoire of type III-secreted effectors for the attaching and effacing pathogens, and provided new insights into the mode of function for LifA/Efa1/ToxB/Z4332, an important family of virulence factors.

Vibrio fluvialis sp. nov. (15) is a halophilic bacterium which was originally isolated in 1975 from a patient with diarrhea from Bahrain from whom no other bacterial enteric pathogens could be isolated (8) and was first called group F (8,11,13,14) or EF-6 (10). V. fluvialis is widely distributed in marine and estuarine environments and has been repeatedly isolated from humans exhibiting severe diarrheal disease (4,8,10,15,17,19,20). The clinical features of the disease closely resemble cholera, except that 75% of the patients examined during a V. fluvialis-associated diarrheal outbreak in Bangladesh in 1976–1977 had blood and mucus in their stools (10). Reports on the enteropathogenicity of this organism have yielded conflicting results ranging from negative reports of enterotoxin production (4, 10, 17, 20) to reports that whole cultures and sterile culture filtrates cause fluid accumulation in rabbit ileal loops as well as cytotoxic and cytotonic responses in tissue culture (1, 19). However, no toxin has been found to be associated with these effects. These observations prompted us to investigate the possibility that V. fluvialis produces toxin(s) which might contribute to the pathogenesis of the disease attributed to this organism.

Classically, pathogenic bacteria are classified as intracellular or extracellular pathogens. Intracellular bacterial pathogens, as Mycobacterium tuberculosis, Salmonella enterica, Brucella suis, or Listeria monocytogenes, can replicate within host cells. After entering the target host cell (professional or non-professional phagocyte), the intracellular pathogen follows vacuolar or cytosolic pathways to replicate. In contrast, extracellular pathogens, as Staphylococcus aureus, Pseudomonas aeruginosa or streptococci, avoid phagocytosis, thus promoting extracellular multiplication. However, the situation appears more complex with a dual lifestyle of intracellular/extracellular bacterial pathogens (Silva, 2012). Indeed, diverse bacterial intracellular pathogens have the ability to produce extracellular infections as a second phase after the initial intracellular stage. Conversely, several extracellular pathogens can enter host cells in vivo, resulting in a phase of intracellular residence, which can be of importance prior the typical extracellular infection. During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses that include oxidative and acid stresses, toxic metal cations, and antimicrobial peptides. Bacterial pathogens have developed strategies to escape the innate immune system and counteract the microbicidal action of macrophages (reviewed by Sarantis and Grinstein, 2012). Whereas some pathogens inhibit phagocytosis (extracellular pathogens), others use virulence factors to subvert the macrophage antimicrobial role and manipulate the host-cell to establish a replication niche. Hence, intracellular pathogens overcome macrophage defenses and use these immune cells as residence and dissemination strategies. Even if it accounts for a transitory initial event, so-called extracellular pathogens can also encounter an intramacrophage stage and have therefore to escape the killing by these immune cells. Intracellular bacteria use a broad range of molecular pathogenicity determinants to manipulate host cell processes and adapt to the intracellular environment. Recent data, described below, reveal that so-called extracellular pathogens have also acquired similar pathogenicity determinants to improve intramacrophage survival.

Polysaccharides are polymers of carbohydrates with anenormous structural diversity, from long linear repetition ofthe same monomer to highly branched structures of dif-ferent sugars. This high structural diversity reflects thefunctional diversity of these molecules. There are twotypes of polysaccharides, storage polysaccharides (i.e.glycogen) and structural polysaccharides, which are nor-mally secreted by the cell and form different cell structures(i.e. cellulose, chitin). Extracellular polysaccharides orexopolysaccharides belong to this last group.Exopolysaccharides are produced not only by microor-ganisms, but also by algae, plants and animals (Suther-land, 2005). Bacterial exopolysaccharides are a majorcomponent of the extracellular polymeric substance(EPS) or matrix of biofilms, and mediate most of thecell-to-cell and cell-to-surface interactions required forbiofilm formation and stabilization (Flemming and Win-gender, 2010). The matrices of biofilms from natural envi-ronments, such as marine and fresh water, soil, or chronicinfections, contain a ubiquitous composition of polysac-charides. More than 30 different matrix polysaccharideshave been characterized so far. Several are homopoly-saccharides (i.e. glucans, fructans, cellulose), but most ofthese are heteropolysaccharides consisting on a mixtureof sugar residues. Exopolysaccharides can even differbetween strains of single species, as exemplified bystrains of

Sea horse (Hippocampus kuda) is one of the ornamental marine organisms and raw material of traditional medicine. Since 1993, Seafarming Development Centre, Lampung has pioneered a research and culture of sea horse in Indonesia. The serious problem in the culture of sea horse is pathogenic bacteria caused death of juveniles and broodstocks. The objective of this study was to identify pathogenic bacteria isolated from sea horse in Seafarming Development Centre. Koch Postulate test was carried out, and then the pathogenic bacteria were identifed by morphological and biochemical tests. Results showed that from a total of 6 bacterial strains isolated from diseased sea horse, 3 strains were pathogenic bacteria to sea horse. These 3 pathogenic bacteria caused identical disease signs with the initial disease signs when the bacteria were isolated. Morphological and biochemical tests suggested that the pathogenic bacteria could be identified to be Vibrio fluvialis, V. alginolyticus and V. hollisae.

Breaking the Stereotype: Virulence Factor–Mediated Protection of Host Cells in Bacterial Pathogenesis

Shigella species are the main cause of bacillary diarrhoea or shigellosis in humans. These organisms are the inhabitants of the human intestinal tract; however, they are one of the main concerns in public health in both developed and developing countries. In this study, we reviewed and summarised the previous studies and recent advances in molecular mechanisms of pathogenesis of Shigella Dysenteriae and non-Dysenteriae species. Regarding the molecular mechanisms of pathogenesis and the presence of virulence factor encoding genes in Shigella strains, species of this bacteria are categorised into Dysenteriae and non-Dysenteriae clinical groups. Shigella species uses attachment, invasion, intracellular motility, toxin secretion and host cell interruption mechanisms, causing mild diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome diseases in humans through the expression of effector delivery systems, protein effectors, toxins, host cell immune system evasion and iron uptake genes. The investigation of these genes and molecular mechanisms can help us to develop and design new methods to detect and differentiate these organisms in food and clinical samples and determine appropriate strategies to prevent and treat the intestinal and extraintestinal infections caused by these enteric pathogens.