Quantification and surface localization of the hemolysin A type 1 secretion system at the endogenous level and under conditions of overexpression

Secretion systems are essential for Gram-negative bacteria as these nanomachineries allow a communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type one secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli (E. coli) , which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC and the substrate HlyA, a member of the family of RTX (repeats in toxins) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression (T7 expression system, BL21(DE3)-BD). The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by super-resolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence the polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS cluster at the outer membrane generating domains of so far not described identity. Importance Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide representing a global burden to the healthcare system. UPEC secrete many virulence factors among these the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the super-resolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

Genotypic Characterization of Pseudomonas aeruginosa Isolates from Eyes with Keratitis and Healthy Conjunctival Sacs

Pseudomonas aeruginosa(P. aeruginosa) was a second most common commensal bacterium in healthy conjunctival sacs. When the corneal epithelial barrier is damaged, P. aeruginosa could cause keratitis, which progresses rapidly and results in corneal perforation and the loss of vision. However, the similarities and differences in characteristics between P. aeruginosa isolates from the eyes with keratitis and those from healthy conjunctival sacs are still poorly understood. In this study, four P. aeruginosa isolates from eyes with keratitis and three P. aeruginosa isolates from healthy conjunctival sacs were obtained, and genotypically characterized using Illumina high-throughput RNA sequencing. A total of 557 differentially expressed genes (DEGs) were found and included 332 upregulated genes and 225 downregulated genes in the keratitis group versus the healthy conjunctival sacs group. Of 557 DEGs, 11 DEGs analyzed with GO enrichment and the KEGG pathway were involved with the bacterial secretion system and pyoverdine metabolism and validated using quantitative reverse-transcription polymerase chain reaction. P. aeruginosa from eyes with keratitis induced more severe corneal infection and higher clinical scores in the mice. These results will contribute to develop alternative therapeutic interventions targeting virulence factors in these DEGs and facilitate the selection of therapeutic strategies.

A Widespread Bacterial Secretion System with Diverse Substrates

The microbial constituency of a host-associated microbiome emerges from a complex physical and chemical interplay of microbial colonization factors, host surface conditions, and host immunological responses. To fill unique niches within a host, bacteria encode surface and secreted proteins that enable interactions with and responses to the host and cooccurring microbes.

Hemolysin Co-regulated Family Proteins Hcp1 and Hcp2 Contribute to Edwardsiella ictaluri Pathogenesis

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. E. ictaluri strain 93–146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double E. ictaluri mutants (EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2) were generated and characterized. Catfish peritoneal macrophages were able to kill EiΔhcp2 better than EiΔevpC, EiΔevpCΔhcp2, and E. ictaluri wild-type (EiWT). The attachment of EiΔhcp2 and EiΔevpCΔhcp2 to ovary cells significantly decreased compared to EiWT whereas the cell invasion rates of these mutants were the same as that of EiWT. Mutants exposed to normal catfish serum in vitro showed serum resistance. The fish challenges demonstrated that EiΔevpC and EiΔevpCΔhcp2 were attenuated completely and provided excellent protection against EiWT infection in catfish fingerlings. Interestingly, EiΔhcp2 caused higher mortality than that of EiWT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with EiΔevpC and EiΔevpCΔhcp2, their attenuation and protection were significantly higher compared to EiWT and sham groups, respectively. Taken together, our data indicated that evpC (hcp1) is involved in E. ictaluri virulence in catfish while hcp2 is involved in adhesion to epithelial cells and survival inside catfish macrophages.

Structure of the mycobacterial ESX-5 type VII secretion system pore complex

The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.

Soil causes gut microbiota to flourish and total serum IgE levels to decrease in mice

Background Traditional farm environments provide protection from allergic diseases. In this study, farm environmental factors were classified into three categories: environmental microbes, soil, and organic matter. To explore the impact of soil and environmental microorganisms on gut microbiota and immune function, mice were fed sterilized soil, soil microbes (in lieu of environmental microbes), or non-sterilized soil. Results Metagenomic sequencing results showed that the intake of sterile soil while inhaling a small amount of soil microbes in the air, increased gut microbial diversity and the abundance of type III secretion system (T3SS) genes and decreased total serum IgE levels induced by 2-4-dinitrofluorobenzene. The intake of soil microbes increased the abundance of genes involved in the metabolism of short-chain fatty acids and amino acid biosynthesis. By contrast, the intake of soil increased gut microbial diversity, the abundance of T3SS genes and related infectious elements, and genes associated with the metabolism of short-chain fatty acids and amino acid biosynthesis and decreased serum IgE levels. The immune function was positively and significantly correlated with the bacterial secretion system genes, especially with that of T3SS. Conclusions An important mechanism through which farm environments exert a protective effect against allergic diseases could be by serving as a “prebiotic” promoting the reproduction and growth of some intestinal microorganisms that harbor bacterial secretion system genes, especially those of T3SS, whose abundance was positively and significantly correlated with innate immune function of mice.

Soil causes gut microbiota to flourish and total serum IgE levels to decrease in mice

BackgroundTraditional farm environments provide protection from allergic diseases. In this study, farm environmental factors were classified into three categories: environmental microbes, soil, and organic matter. To explore the impact of soil and environmental microorganisms on gut microbiota and immune function, mice were fed sterilized soil, soil microbes (in lieu of environmental microbes), or non-sterilized soil.ResultsMetagenomic sequencing results showed that the intake of sterile soil while inhaling a small amount of soil microbes in the air, increased gut microbial diversity and the abundance of type III secretion system (T3SS) genes and decreased total serum IgE levels induced by 2-4-dinitrofluorobenzene. The intake of soil microbes increased the abundance of genes involved in the metabolism of short-chain fatty acids and amino acid biosynthesis. By contrast, the intake of soil increased gut microbial diversity, the abundance of T3SS genes and related infectious elements, and genes associated with the metabolism of short-chain fatty acids and amino acid biosynthesis and decreased serum IgE levels. The immune function was positively and significantly correlated with the bacterial secretion system genes, especially with that of T3SS.ConclusionsAn important mechanism through which farm environments exert a protective effect against allergic diseases could be by serving as a “prebiotic” promoting the reproduction and growth of some intestinal microorganisms that harbor bacterial secretion system genes, especially those of T3SS, whose abundance was positively and significantly correlated with innate immune function of mice.

Review of Potential Pseudomonas Weaponry, Relevant to the Pseudomonas–Aspergillus Interplay, for the Mycology Community

Pseudomonas aeruginosa is one of the most prominent opportunistic bacteria in airways of cystic fibrosis patients and in immunocompromised patients. These bacteria share the same polymicrobial niche with other microbes, such as the opportunistic fungus Aspergillus fumigatus. Their inter-kingdom interactions and diverse exchange of secreted metabolites are responsible for how they both fare in competition for ecological niches. The outcomes of their contests likely determine persistent damage and degeneration of lung function. With a myriad of virulence factors and metabolites of promising antifungal activity, P. aeruginosa products or their derivatives may prove useful in prophylaxis and therapy against A. fumigatus. Quorum sensing underlies the primary virulence strategy of P. aeruginosa, which serves as cell–cell communication and ultimately leads to the production of multiple virulence factors. Understanding the quorum-sensing-related pathogenic mechanisms of P. aeruginosa is a first step for understanding intermicrobial competition. In this review, we provide a basic overview of some of the central virulence factors of P. aeruginosa that are regulated by quorum-sensing response pathways and briefly discuss the hitherto known antifungal properties of these virulence factors. This review also addresses the role of the bacterial secretion machinery regarding virulence factor secretion and maintenance of cell–cell communication.

Nascent SecM chain interacts with outer ribosomal surface to stabilize translation arrest

SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57–98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57–98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.