Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the ...formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.
The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic ...effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila . We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.
How different microbial species compete for specific niches is not fully understood. Here, we focus on the type VI secretion system (T6SS), a specialized protein delivery system, which many Gram-negative bacteria use to kill eukaryotic and prokaryotic competitors by translocating toxic protein molecules to target cells. Identification of effectors is required for understanding the pivotal role that the T6SS plays in dictating interbacterial and bacterialâhost dynamics. In this study, we describe a new approach to systematically identify T6SS effectors. We also demonstrate that secretion of effectors requires interaction with a set of cognate effector-binding chaperone proteins that we define in this study, providing important insights for understanding the mechanism of T6SS effector delivery.
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Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although ...widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.
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Pseudomonas syringae employs a type III secretion system to inject 20-30 different type III effector (T3SE) proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses ...and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found in both plant and animal pathogenic bacteria. In P. syringae, this superfamily includes the evolutionarily diverse HopZ1, HopZ2 and HopZ3 alleles. To investigate the roles of the HopZ family in immunomodulation, we generated dexamethasone-inducible T3SE transgenic lines of Arabidopsis for HopZ family members and characterized them for immune suppression phenotypes. We show that all of the HopZ family members can actively suppress various facets of Arabidopsis immunity in a catalytic residue-dependent manner. HopZ family members can differentially suppress the activation of mitogen-activated protein (MAP) kinase cascades or the production of reactive oxygen species, whereas all members can promote the growth of non-virulent P. syringae. Localization studies show that four of the HopZ family members containing predicted myristoylation sites are localized to the vicinity of the plasma membrane while HopZ3 which lacks the myristoylation site is at least partially nuclear localized, suggesting diversification of immunosuppressive mechanisms. Overall, we demonstrate that despite significant evolutionary diversification, all HopZ family members can suppress immunity in Arabidopsis.
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Academic conferences are integral to the dissemination of novel research findings and discussion of pioneering ideas across all postsecondary disciplines. For some participants, these environments ...are spaces to develop new collaborations, research projects, and social bonds; however, for others, conferences can be a place of marginalization and outright hostility. To assess how diverse individuals experience conference spaces, we interpreted results from a conference climate survey filled out by 198 of 482 registrants of the Society for the Advancement of Biology Education Research (SABER) West 2021 conference. Analysis of the survey data was conducted by six biology education researchers, who in addition to raising conference participant voices, provide insights, and next steps whose implementation can promote greater participant equity, representation, and engagement in future science, technology, engineering, and math (STEM) education conferences specifically and potentially all academic conference spaces more broadly.
The Gram negative bacterial phytopathogen Pseudomonas syringae employs a molecular syringe termed the Type III secretion system (TTSS) to deliver an array of Type III secreted effector (TTSE) ...proteins into plant cells. The major function ascribed to type III effectors of P. syringae is their ability to suppress plant immunity. Because individual pathovars of P. syringae can possess over 30 TTSEs, functional redundancy can provide a hurdle to ascribing functions by TTSE-deletion or -overexpression in such TTSE-rich backgrounds. Approaches to overcome functional redundancy have included the deletion of multiple TTSEs from individual pathovars as well as engineering the plant commensal P. fluorescens strain to express the P. syringae TTSS and deliver P. syringae TTSEs. As we describe here, transgenic Arabidopsis plants expressing individual TTSEs have also be used to overcome problems of functional redundancy and provide invaluable insights into TTSE virulence functions.
Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes ...that serve as "non-self" features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as "non-self" features or induce a "modified-self" state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2 Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2 Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2 Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2 Pto . In support of this hypothesis, HopF2 Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2 Pto did not induce ETI and instead promoted P. syringae growth in ARABIDOPSIS: This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2 Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.
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