GacS/GacA is a conserved two‐component system that functions as a master regulator of virulence‐associated traits in many bacterial pathogens, including
- Award ID(s):
- 1942898
- NSF-PAR ID:
- 10271600
- Date Published:
- Journal Name:
- Microorganisms
- Volume:
- 9
- Issue:
- 6
- ISSN:
- 2076-2607
- Page Range / eLocation ID:
- 1227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA‐regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogenPseudomonas aeruginosa , GacS/GacA negatively regulates the expression of type III secretion system (T3SS)‐encoding genes. However, in the plant pathogenic bacteriumPseudomonas syringae , strain‐to‐strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re‐evaluated the function of GacA inP. syringae pv.tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence insideArabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface‐inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulatingP. syringae virulence. -
Burbank, Lindsey Price (Ed.)
ABSTRACT Type VI secretion system (T6SS) is a versatile, contact-dependent contractile nano-weapon in Gram-negative bacteria that fires proteinaceous effector molecules directly into prokaryotic and eukaryotic cells aiding in manipulation of the host and killing of competitors in complex niches. In plant pathogenic xanthomonads, T6SS has been demonstrated to play these diverse roles in individual pathosystems. However, the molecular network underlying the regulation of T6SS is still elusive in
Xanthomonas spp. To bridge this knowledge gap, we conducted anin vitro transcriptome screen using plant apoplast mimicking minimal medium, XVM2 medium, to decipher the effect oftssM deletion, a core gene belonging to T6SS-cluster i3*, on the regulation of gene expression inXanthomonas perforans strain AL65. Transcriptomic data revealed that a total of 277 and 525 genes were upregulated, while 307 and 392 genes were downregulated in the mutant strain after 8 and 16 hours of growth in XVM2 medium. The transcript abundance of several genes associated with flagellum and pilus biogenesis as well as type III secretion system was downregulated in the mutant strain. Deletion oftssM of cluster-i3* resulted in upregulation of several T6SS genes belonging to cluster-i3*** and genes involved in biofilm and cell wall biogenesis. Similarly, transcription regulators likerpoN , Pho regulon,rpoE , andcsrA were identified to be upregulated in the mutant strain. Our results suggest that T6SS modulates the expression of global regulators likecsrA ,rpoN , andpho regulons, triggering a signaling cascade, and co-ordinates the expression of suite of virulence factors, stress response genes, and metabolic genes.IMPORTANCE T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here,
Xanthomonas . However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* inX. perforans AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria. -
Abstract Agrobacterium -mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties toAgrobacterium infection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineeringAgrobacterium tumefaciens to express a type III secretion system (T3SS) fromPseudomonas syringae and individually deliver theP. syringae effectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineeredAgrobacterium , we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%–400%. We also show that engineeredA. tumefaciens expressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner. -
Abstract Like many Gram‐negative pathogens,
Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle‐like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo‐hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high‐resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation ofShigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulatesShigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross‐species therapeutic that broadly targets T3SS ATPase oligomerization and activation. -
The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, including NF-κβ (c-rel and relB), creb3L4, socs6 and foxo3a. Regulation of transcription factors leads to regulation of proinflammatory interleukins (IL-8, IL-12a, IL-15, IL-6) and cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 mRNA by WT E. ictaluri leads to decreased production of prostaglandin E2 (PGE2), which is the product of COX-2 activity. Collectively, our results indicate that E. ictaluri EseN is an important player in the modulation of host immune responses to E.ictaluri infection.more » « less