Killing with proficiency:integrated post-translational regulation of an offensive Type VI secretion system

<div><p>The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. ‘Defensive’ T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are ‘offensive’, firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of <i>Serratia marcescens</i> to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in <i>S</i>. <i>marcescens</i>. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.</p></div

The prevalence and origin of exoprotease-producing cells in the <em>Bacillus subtilis </em>biofilm

Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway(1,2). Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis

Aim, Load, Fire:The Type VI Secretion System, a Bacterial Nanoweapon

International audienc

Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis

Mitochondrial dynamics and functionality are linked to the autophagic degradative pathway under several stress conditions. However, the interplay between mitochondria and autophagy upon cell death signalling remains unclear. The T-cell receptor pathway signals the so-called activation-induced cell death (AICD) essential for immune tolerance regulation. Here, we show that this apoptotic pathway requires the inhibition of macroautophagy. Protein kinase-A activation downstream of T-cell receptor signalling inhibits macroautophagy upon AICD induction. This leads to the accumulation of damaged mitochondria, which are fragmented, display remodelled cristae and release cytochrome c, thereby driving apoptosis. Autophagy-forced reactivation that clears the Parkin-decorated mitochondria is as effective in inhibiting apoptosis as genetic interference with cristae remodelling and cytochrome c release. Thus, upon AICD induction regulation of macroautophagy, rather than selective mitophagy, ensures apoptotic progression

Rhs1 and Rhs2 depend exclusively on VgrG2 for their delivery into target cells.

<p>(A-B) Recovery of a target strain lacking <i>rhsI1</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>tssH</i>Δ<i>rhsI1</i>), part A, or lacking <i>rhsI2</i> (<i>S</i>. <i>marcescens</i> Db10 Δ<i>rhs2</i>Δ<i>rhsI2</i>), part B, following co-culture with wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>rhs1</i>, Δ<i>rhs2</i>, Δ<i>vgrG1</i>, Δ<i>vgrG2</i> and Δ<i>vgrG1</i>Δ<i>vgrG2</i>) strains of Db10 as attacker, as indicated. Points show mean ± SEM (n = 4).</p

VgrG2 requires at least one of its specific PAAR proteins for secretion but not stability.

<p>(A) A VgrG2-His₆ fusion protein (VgrG2-His) encoded at the normal chromosomal location is fully functional, whereas VgrG1-His₆ (VgrG1-His) is non-functional. Recovery of target organism <i>P</i>. <i>fluorescens</i> 55 following co-culture with wild type <i>S</i>. <i>marcescens</i> Db10 (WT), the Δ<i>tssE</i>, Δ<i>vgrG1</i> and Δ<i>vgrG2</i> mutants, or strains expressing VgrG2-His in the Δ<i>vgrG1</i> background or VgrG1-His in the Δ<i>vgrG2</i> background. Points show mean +/-SEM (n = 4). (B) Anti-His immunoblot of cellular and secreted protein from wild type <i>S</i>. <i>marcescens</i> Db10 or strains expressing the chromosomal VgrG2-His₆ fusion protein, either in a wild type background (VgrG2-His) or in strains lacking TssE (VgrG2-His, Δ<i>tssE</i>), Rhs1 (VgrG2-His, Δ<i>rhs1</i>), Rhs2 (VgrG2-His, Δ<i>rhs2</i>) or both Rhs proteins (VgrG2-His, Δ<i>rhs1</i>Δ<i>rhs2</i>). (C) Immunoblot detection of VgrG2-His in the cellular and secreted fractions of strains expressing VgrG2-His in a wild type or Δ<i>rhs1</i>Δ<i>rhs2</i> background and carrying either the vector control plasmid (+VC) or plasmids directing the expression of Rhs1 and RhsI1 (+Rhs1, pSC791) or Rhs2 and RhsI2 (+Rhs2, pSC788) <i>in trans</i>. The empty vector control for Rhs2 was pSUPROM; for Rhs1 it was pBAD18-Kn and expression was induced with 0.0002% l-arabinose.</p