Structural characterization and functional insights into the type II secretion system of the poly-extremophile Deinococcus radiodurans.

The extremophile bacterium D. radiodurans boasts a distinctive cell envelope characterized by the regular arrangement of three protein complexes. Among these, the Type II Secretion System (T2SS) stands out as a pivotal structural component. We used cryo-electron microscopy to reveal unique features, such as an unconventional protein belt (DR_1364) around the main secretin (GspD), and a cap (DR_0940) found to be a separated subunit rather than integrated with GspD. Furthermore, a novel region at the N-terminus of the GspD constitutes an additional second gate, supplementing the one typically found in the outer membrane region. This T2SS was found to contribute to envelope integrity, while also playing a role in nucleic acid and nutrient trafficking. Studies on intact cell envelopes show a consistent T2SS structure repetition, highlighting its significance within the cellular framework.

The type II secretion system as an underappreciated and understudied mediator of interbacterial antagonism.

Interbacterial antagonism involves all major phyla, occurs across the full range of ecological niches, and has great significance for the environment, clinical arena, and agricultural and industrial sectors. Though the earliest insight into interbacterial antagonism traces back to the discovery of antibiotics, a paradigm shift happened when it was learned that protein secretion systems (e.g., types VI and IV secretion systems) deliver toxic "effectors" against competitors. However, a link between interbacterial antagonism and the Gram-negative type II secretion system (T2SS), which exists in many pathogens and environmental species, is not evident in prior reviews on bacterial competition or T2SS function. A current examination of the literature revealed four examples of a T2SS or one of its known substrates having a bactericidal activity against a Gram-positive target or another Gram-negative. When further studied, the T2SS effectors proved to be peptidases that target the peptidoglycan of the competitor. There are also reports of various bacteriolytic enzymes occurring in the culture supernatants of some other Gram-negative species, and a link between these bactericidal activities and T2SS is suggested. Thus, a T2SS can be a mediator of interbacterial antagonism, and it is possible that many T2SSs have antibacterial outputs. Yet, at present, the T2SS remains relatively understudied for its role in interbacterial competition. Arguably, there is a need to analyze the T2SSs of a broader range of species for their role in interbacterial antagonism. Such investigation offers, among other things, a possible pathway toward developing new antimicrobials for treating disease.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Transcriptome Analysis Reveals Cross-Talk between the Flagellar Transcriptional Hierarchy and Secretion System in Plesiomonas shigelloides.

Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides' coordinated survival in the natural environment and the mechanisms that infect the host.

Acetic and citric acids effect the type II secretion system and decrease the metabolic activities of salmon spoilage-related Rahnella aquatilis KM05.

Rahnella aquatilis causes seafoods to spoil by metabolizing sulfur-containing amino acids and/or proteins, producing H2S in products. The type II secretion system (T2SS) regulates the transport of proteases from the cytoplasm to the surrounding environment and promotes bacterial growth at low temperatures. To prevent premature fish spoilage, new solutions for inhibiting the T2SS of bacteria should be researched. In this study, global transcriptome sequencing was used to analyze the spoilage properties of R. aquatilis KM05. Two of the mapped genes/coding sequences (CDSs) were matched to the T2SS, namely, qspF and gspE, and four of the genes/CDSs, namely, ftsH, rseP, ptrA and pepN, were matched to metalloproteases or peptidases in R. aquatilis KM05. Subinhibitory concentrations of citric (18 µM) and acetic (41 µM) acids caused downregulation of T2SS-related genes (range from - 1.0 to -4.5) and genes involved in the proteolytic activities of bacteria (range from - 0.5 to -4.0). The proteolytic activities of R. aquatilis KM05 in vitro were reduced by an average of 40%. The in situ experiments showed the antimicrobial properties of citric and acetic acids against R. aquatilis KM05; the addition of an acidulant to salmon fillets limited microbial growth. Citric and acetic acids extend the shelf life of fish-based products and prevent food waste.

Unraveling extracellular protein signatures to enhance live attenuated vaccine development through type II secretion system disruption in Vibriomimicus.

Type II secretion systems (T2SS) are important molecular machines used by bacteria to transport a wide range of proteins across the outer membrane from the periplasm. Vibrio mimicus is an epidemic pathogen threats to both aquatic animals and human health. Our previous study demonstrates that T2SS deletion reduced virulence by 307.26 times in yellow catfish. However, the specific effects of T2SS-mediated extracellular protein secretion in V. mimicus, including its potential role in exotoxin secretion or other mechanisms, require further investigation. Through proteomics and phenotypic analyses, this study observed that the ΔT2SS strain exhibited significant self-aggregation and dynamic deficiency, with a notable negative correlation with subsequent biofilm formation. The proteomics analysis revealed 239 different abundances of extracellular proteins after T2SS deletion, including 19 proteins with higher abundance and 220 proteins with lower and even absent in the ΔT2SS strain. These extracellular proteins are involved in various pathways, such as metabolism, virulence factors expression, and enzymes. Among them, purine, pyruvate, and pyrimidine metabolism, and the Citrate cycle, were the primary pathways affected by T2SS. Our phenotypic analysis is consistent with these findings, suggesting that the decreased virulence of ΔT2SS strains is due to the effect of T2SS on these proteins, which negatively impacts growth, biofilm formation, auto-aggregation, and motility of V. mimicus. These results provide valuable insights for designing deletion targets for attenuated vaccines development against V. mimicus and expand our understanding of the biological functions of T2SS.

Comparative Transcriptomic Analysis of Wheat Cultivars in Response to Xanthomonas translucens pv. cerealis and Its T2SS, T3SS, and TALEs Deficient Strains.

Xanthomonas translucens pv. cerealis causes bacterial leaf streak disease on small grain cereals. Type II and III secretion systems (T2SS and T3SS) play a pivotal role in the pathogenicity of the bacterium, while no data are available on the transcriptomic profile of wheat cultivars infected with either wild type (WT) or mutants of the pathogen. In this study, WT, TAL-effector mutants, and T2SS/T3SS mutants of X. translucens pv. cerealis strain NXtc01 were evaluated for their effect on the transcriptomic profile of two wheat cultivars, 'Chinese Spring' and 'Yangmai-158', using Illumina RNA-sequencing technology. RNA-Seq data showed that the number of differentially expressed genes (DEGs) was higher in Yangmai-158 than in Chinese Spring, suggesting higher susceptibility of Yangmai-158 to the pathogen. In T2SS, most suppressed DEGs were related to transferase, synthase, oxidase, WRKY, and bHLH transcription factors. The gspD mutants showed significantly decreased disease development in wheat, suggesting an active contribution of T2SS in virulence. Moreover, the gspD mutant restored full virulence and its multiplication in planta by addition of gspD in trans. In the T3SS-deficient strain, downregulated DEGs were associated with cytochrome, peroxidases, kinases, phosphatases, WRKY, and ethylene-responsive transcription factors. In contrast, upregulated DEGs were trypsin inhibitors, cell number regulators, and calcium transporter. Transcriptomic analyses coupled with quantitative real-time-PCR indicated that some genes are upregulated in Δtal1/Δtal2 compared with the tal-free strain, but no direct interaction was observed. These results provide novel insight into wheat transcriptomes in response to X. translucens infection and pave the way for understanding host-pathogen interactions.

A novel strain of Shigella isolated from the gut of Lepidocephalichthys guntea has in its genome a complete gene package for Type ll secretion system, and elaborate repertoire of genes responsible for multiple antibiotic-resistance and metal resistance via specific efflux channels.

The bacterial strain GCP5 was isolated from the gut of a bottom-dwelling fish Lepidocephalichthys guntea, that lives in the Magurmari River near North Bengal University in Siliguri, India. GCP5 was phylogenetically assigned to the Shigella genus using whole genome-based trees, k-mer analysis, the multilocus species tree (MLST), and single nucleotide polymorphism (SNP)-based trees, and the genetic makeup of the isolate was determined following assembly of the genome sequences and genome annotation with several bioinformatics tools. The presence of a complete package of general-secretory-pathway (gsp) genes, grouped in an operon identical to a well-characterized type II secretion system (T2SS), was confirmed by genome mining of Shigella sp. GCP5. The operon's gsp genes shared the most homology with Escherichia coli gsp genes. A few more high-pathogenicity islands (HPIs) in the GCP5 genome were validated using the pan-genomes analysis pipeline (PGAP) and island viewer. Several antibiotic-resistance genes were found in this genome, as well as the existence of key antibiotic efflux pump families, allowing for the creation of a gene network of several antibiotic efflux transporters. In addition, the genome contained genes specific for nickel transport, the nikABCD system, and the RND family transporter cusCFBA, which confers resistance to copper and silver by effluxing out Cu+ and Ag+ ions.

Structure-function analysis of pectate lyase Pel3 reveals essential facets of protein recognition by the bacterial type 2 secretion system.

The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS.

RpoN is required for the motility and contributes to the killing ability of Plesiomonas shigelloides.

BACKGROUND: RpoN, also known as σ54, first reported in Escherichia coli, is a subunit of RNA polymerase that strictly controls the expression of different genes by identifying specific promoter elements. RpoN has an important regulatory function in carbon and nitrogen metabolism and participates in the regulation of flagellar synthesis, bacterial motility and virulence. However, little is known about the effect of RpoN in Plesiomonas shigelloides. RESULTS: To identify pathways controlled by RpoN, RNA sequencing (RNA-Seq) of the WT and the rpoN deletion strain was carried out for comparison. The RNA-seq results showed that RpoN regulates ~ 13.2% of the P. shigelloides transcriptome, involves amino acid transport and metabolism, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, ribosome biosynthesis, flagellar assembly and bacterial secretion system. Furthermore, we verified the results of RNA-seq using quantitative real-time reverse transcription PCR, which indicated that the absence of rpoN caused downregulation of more than half of the polar and lateral flagella genes in P. shigelloides, and the ΔrpoN mutant was also non-motile and lacked flagella. In the present study, the ability of the ΔrpoN mutant to kill E. coli MG1655 was reduced by 54.6% compared with that of the WT, which was consistent with results in RNA-seq, which showed that the type II secretion system (T2SS-2) genes and the type VI secretion system (T6SS) genes were repressed. By contrast, the expression of type III secretion system genes was largely unchanged in the ΔrpoN mutant transcriptome and the ability of the ΔrpoN mutant to infect Caco-2 cells was also not significantly different compared with the WT. CONCLUSIONS: We showed that RpoN is required for the motility and contributes to the killing ability of P. shigelloides and positively regulates the T6SS and T2SS-2 genes.

The structure of Acinetobacter-secreted protease CpaA complexed with its chaperone CpaB reveals a novel mode of a T2SS chaperone-substrate interaction.

Members of the Acinetobacter baumannii-calcoaceticus complex are nosocomial pathogens frequently causing multidrug-resistant infections that are increasing at alarming rates. A. baumannii has become the Gram-negative bacterium with the highest rate of multidrug resistance. As such, it is categorized by the World Health Organization as a critical priority for the research and development of new antimicrobial therapies. The zinc-dependent metalloendopeptidase CpaA is a predominant substrate of the type II secretion system (T2SS). CpaA is also a virulence factor of medically relevant Acinetobacter strains that specifically degrade the human glycoprotein coagulation factor XII and not its deglycosylated form, but the mechanism for this specificity is unknown. CpaB is a membrane-anchored T2SS chaperone that interacts with CpaA and is required for its stability and secretion. Here, we report the crystal structure of the CpaAB complex at 2.6-Å resolution, revealing four glycan-binding domains in CpaA that were not predicted from its primary sequence and may explain CpaA's glycoprotein-targeting activity. The structure of the complex identified a novel mode for chaperone-protease interactions in which the protease surrounds the chaperone. The CpaAB organization was akin to zymogen inactivation, with CpaB serving as a prodomain that inhibits catalytically active CpaA. CpaB contains a C-terminal tail that appears to block access to the CpaA catalytic site, and functional experiments with truncated variants indicated that this tail is dispensable for CpaA expression and secretion. Our results provide new insight into the mechanism of CpaA secretion and may inform the future development of therapeutic strategies for managing Acinetobacter infections.

Differential Modulation of Quorum Sensing Signaling through QslA in Pseudomonas aeruginosa Strains PAO1 and PA14.

Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite the isolates being closely related, PA14 exhibits increased virulence compared to that of PAO1 in various models. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected a transcriptomic approach through RNA sequencing. We found 2,029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by quorum sensing (QS), known to control most of the virulence factors in P. aeruginosa Among them, we chose to focus our study on QslA, an antiactivator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hypervirulence phenotype. After confirming that QslA protein was highly produced in PAO1 but not in the PA14 strain, we obtained evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics than PA14. Moreover, known virulence factors activated by QS, such as (i) pyocyanin production, (ii) H2-T6SS (type VI secretion system) gene expression, and (iii) Xcp-T2SS (type II secretion system) machinery production and secretion, were all lower in PAO1 than in PA14, due to higher qslA expression. However, biofilm formation and cytotoxicity toward macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Together, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginosa strains PAO1 and PA14.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen responsible for acute nosocomial infections and chronic pulmonary infections. P. aeruginosa strain PA14 is known to be hypervirulent in different hosts. Despite several studies in the field, the underlining molecular mechanisms sustaining this phenotype remain enigmatic. Here we provide evidence that the PA14 strain has faster quorum sensing (QS) kinetics than the PAO1 strain, due to the lack of QslA expression, an antiactivator of QS. QS is a major regulator of virulence factors in P. aeruginosa; therefore, we propose that the hypervirulent phenotype of the PA14 strain is, at least partially, due to the lack of QslA expression. This mechanism could be of great importance, as it could be conserved among other P. aeruginosa isolates.

Direct interactions between the secreted effector and the T2SS components GspL and GspM reveal a new effector-sensing step during type 2 secretion.

In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpYL and XcpZM, of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa Competition experiments indicated that CbpD binding to XcpYL is XcpZM-dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZM homodimerization and increases the affinity between XcpYL and XcpZM The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding.

Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae.

It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

Dynamics of a type 2 secretion system pseudopilus unraveled by complementary approaches.

Secretion pili, bacterial fibers responsible for transporting proteins to the extracellular milieu in some secretion systems, are very strong structures but at the same time highly flexible. Their flexibility and helical symmetry make structure determination at atomic resolution a challenging task. We have previously used an integrative structural biology approach including liquid-state NMR, cryo-electron microscopy (cryo-EM), and modeling to determine the pseudo-atomic resolution structure of the type 2 secretion system pseudopilus in a mutant form, where we employed NMR to determine the high resolution structure of the pilin (the monomer building block of the pilus). In this work, we determine the pseudo-atomic structure of the wild type pilus, and compare the dynamics of wild type and mutant pili by normal mode analysis. We present a detailed NMR analysis of the dynamics of the pilin in isolation, and compare dynamics and solvent accessibility of isolated and assembled pilins by Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS). These complementary approaches provide a comprehensive view of internal and overall dynamics of pili, crucial for their function.

Comparative proteomics analyses of Acinetobacter baumannii strains ATCC 17978 and AB5075 reveal the differential role of type II secretion system secretomes in lung colonization and ciprofloxacin resistance.

Acinetobacter baumannii is an emerging nosocomial pathogen with alarming antibiotic resistance profiles. A better understanding of the virulence and resistance mechanisms of this pathogen is necessary for identifying new methods to combat its infections in a more efficient way. In this regard, the type II secretion system (T2SS) of A. baumannii is an attractive target majorly secreting lipid-metabolizing enzymes and contributes significantly to its virulence. No attempts have been made to study the differential role, and the nature of T2SS secreted proteins among different strains of A. baumannii. In this study, we compare T2SS substrates and functions between A. baumannii strains ATCC 17978, and the MDR highly virulent strain AB5075. The functional categories of the T2-secreted proteins were analyzed, and the virulence potential of the tested strains was compared in vivo using a murine pneumonia model. Biofilm formation was compared using crystal violet assay in micro-titer plates. The contribution to antibiotic resistance was measured by determining the minimum inhibitory concentration (MIC) of different classes of antibiotic. Results indicate that the T2SS secretome gives a colonization advantage to AB5075 over ATCC 1797 but is more important for biofilm formation by the latter. Transposon insertional inactivation of the general secretory pathway protein D (gspD), which is a key component in the structure of the T2SS, significantly increased the MIC of AB5075 to ciprofloxacin. Our report is the first to describe the strain-dependent evolution of the T2SS secretome in relation to the virulence and antibiotic resistance attributes of Gram-negative species.

Multiple Structures Disclose the Secretins' Secrets.

Bacterial secretins are outer membrane proteins that provide a path for secreted proteins to access the cell exterior/surface. They are one of the core components of secretion machines and are found in type II and type III secretion systems (T2SS and T3SS, respectively). The secretins comprise giant ring-shaped homo-oligomers whose precise atomic organization was only recently deciphered thanks to spectacular developments in cryo-electron microscopy (cryo-EM) imaging techniques.

Natural Transformation in Vibrio parahaemolyticus: a Rapid Method To Create Genetic Deletions.

The Gram-negative bacterium Vibrio parahaemolyticus is an opportunistic human pathogen and the leading cause of seafood-borne acute gastroenteritis worldwide. Recently, this bacterium was implicated as the etiologic agent of a severe shrimp disease with consequent devastating outcomes to shrimp farming. In both cases, acquisition of genetic material via horizontal transfer provided V. parahaemolyticus with new virulence tools to cause disease. Dissecting the molecular mechanisms of V. parahaemolyticus pathogenesis often requires manipulating its genome. Classically, genetic deletions in V. parahaemolyticus are performed using a laborious, lengthy, multistep process. Here, we describe a fast and efficient method to edit this bacterium's genome based on V. parahaemolyticus natural competence. Although this method is similar to one previously described, V. parahaemolyticus requires counterselection for curing of acquired plasmids due to its recalcitrant nature of retaining extrachromosomal DNA. We believe this approach will be of use to the Vibrio community.IMPORTANCE Spreading of vibrios throughout the world correlates with increased global temperatures. As they spread, they find new niches in which to survive, proliferate, and invade. Therefore, genetic manipulation of vibrios is of the utmost importance for studying these species. Here, we have delineated and validated a rapid method to create genetic deletions in Vibrio parahaemolyticus This study provides insightful methodology for studies with other Vibrio species.

Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species.

Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are a frequent cause of multidrug-resistant, healthcare-associated infections. Our previous work demonstrated that A. nosocomialis M2 possesses a functional type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which has been shown previously to cleave human Factor V and deregulate blood coagulation, as the most abundant type II secreted effector protein. We also demonstrated that its secretion is dependent on CpaB, a membrane-bound chaperone. In this study, we show that CpaA expression and secretion are conserved across several medically relevant Acinetobacter species. Additionally, we demonstrate that deletion of cpaA results in attenuation of A. nosocomialis M2 virulence in moth and mouse models. The virulence defects resulting from the deletion of cpaA were comparable with those observed upon abrogation of T2SS activity. The virulence defects resulting from the deletion of cpaA are comparable with those observed upon abrogation of T2SS activity. We also show that CpaA and CpaB strongly interact, forming a complex in a 1:1 ratio. Interestingly, deletion of the N-terminal transmembrane domain of CpaB results in robust secretion of CpaA and CpaB, indicating that the transmembrane domain is dispensable for CpaA secretion and likely functions to retain CpaB inside the cell. Limited proteolysis of spheroplasts revealed that the C-terminal domain of CpaB is exposed to the periplasm, suggesting that this is the site where CpaA and CpaB interact in vivo Last, we show that CpaB does not abolish the proteolytic activity of CpaA against human Factor V. We conclude that CpaA is, to the best of our knowledge, the first characterized, bona fide virulence factor secreted by Acinetobacter species.

Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.