The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells

The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non‐phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin‐driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium‐ and phospholipid‐binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS‐mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho‐family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.     

Identification of Chlamydia trachomatis CT621, a protein delivered through the type III secretion system to the host cell cytoplasm and nucleus

Chlamydiae are obligate intracellular bacteria, developing inside host cells within chlamydial inclusions. From these inclusions, the chlamydiae secrete proteins into the host cell cytoplasm. A pathway through which secreted proteins can be delivered is the type III secretion system (T3SS). The T3SS is common to several gram-negative bacteria and the secreted proteins serve a variety of functions often related to the modulation of host signalling. To identify new potentially secreted proteins, the cytoplasm was extracted from Chlamydia trachomatis L2-infected HeLa cells, and two-dimensional polyacrylamide gel electrophoresis profiles of [³⁵S]-labelled chlamydial proteins from this extract were compared with profiles of chlamydial proteins from the lysate of infected cells. In this way, CT621 was identified. CT621 is a member of a family of proteins containing a domain of unknown function DUF582 that is only found within the genus Chlamydia . Immunofluorescence microscopy and immunoblotting demonstrated that CT621 is secreted late in the chlamydial developmental cycle and that it is the first chlamydial protein found to be localized within both the host cell cytoplasm and the nucleus. To determine whether CT621 is secreted through the T3SS, an inhibitor of this apparatus was added to the infection medium, resulting in retention of the protein inside the chlamydiae. Hence, the so far uncharacterized CT621 is a new type III secretion effector protein.     

Domain Analyses Reveal That Chlamydia trachomatis CT694 Protein Belongs to the Membrane-localized Family of Type III Effector Proteins

The Chlamydia trachomatis type three-secreted effector protein CT694 is expressed during late-cycle development yet is secreted by infectious particles during the invasion process. We have previously described the presence of at least two functional domains within CT694. CT694 was found to interact with the human protein Ahnak through a C-terminal domain and affect formation of host-cell actin stress fibers. Immunolocalization analyses of ectopically expressed pEGFP-CT694 also revealed plasma membrane localization for CT694 that was independent of Ahnak binding. Here we provide evidence that CT694 contains multiple functional domains. Plasma membrane localization and CT694-induced alterations in host cell morphology are dependent on an N-terminal domain. We demonstrate that membrane association of CT694 is dependent on a domain resembling a membrane localization domain (MLD) found in anti-host proteins from Yersinia, Pseudomonas, and Salmonella spp. This domain is necessary and sufficient for localization and morphology changes but is not required for Ahnak binding. Further, the CT694 MLD is able to complement ExoS ΔMLD when ectopically expressed. Taken together, our data indicate that CT694 is a multidomain protein with the potential to modulate multiple host cell processes.     

PipB2 is a substrate of the Salmonella pathogenicity island 1-encoded type III secretion system

Salmonella harbors two type III secretion systems, T3SS1 and T3SS2, encoded on the pathogenicity islands SPI1 and SPI2, respectively. Several effector proteins are secreted through these systems into the eukaryotic host cells. PipB2 is a T3SS2 effector that contributes to the modulation of kinesin-1 motor complex activity. Here, we show that PipB2 is also a substrate of T3SS1. This result was obtained infecting human epithelial HeLa cells for 2 h and was confirmed in murine RAW264.7 macrophages, and rat NRK fibroblasts. Analysis at different time points after infection revealed that translocation of PipB2 is T3SS1-dependent in epithelial cells throughout the infection. In contrast, translocation into macrophages is T3SS1-dependent during invasion but T3SS2-dependent at later time points. The N-terminal 10 amino acid residues contain the signal necessary for translocation through both systems. These results confirm the functional overlap between these virulence-related secretion systems and suggest a new role for the effector PipB2.     

Structural characterization of a novel Chlamydia pneumoniae type III secretion-associated protein, Cpn0803

Type III secretion (T3S) is an essential virulence factor used by gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S.     

Identification of Anaplasma marginale type IV secretion system effector proteins

Background

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.

Results

By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.

Conclusions

The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.     

Multiple lessons from the multiple functions of a regulator of type III secretion system assembly in the plant pathogen Pseudomonas syringae

The assembly of type III secretion systems (T3SSs), which inject bacterial effector proteins into the cytosol of animal and plant hosts, is a highly regulated process. Animal pathogens use a length-control protein to produce T3SS needles of fixed length and then a second regulator, such as YopN in Yersinia spp, to mediate host contact-dependent effector delivery. For Pseudomonas syringae and other plant pathogens, regulation of the assembly process differs because the T3SS pilus must grow through variably thick plant cell walls before contacting the host plasma membrane. In this issue of Molecular Microbiology, Crabill et al. (2012) report evidence that the YopN homologue HrpJ is a multifunctional regulator of T3SS assembly in DC3000. A hrpJ mutant hyper-secretes pilus protein and no longer secretes four translocator proteins in culture, and it fails to inject effectors in planta. As with other proteins in this class, HrpJ is itself a T3SS substrate, but secretion-incompetent forms retain regulatory function. However, HrpJ is unusual in suppressing innate immune responses within host cells, as demonstrated with transgenic plants. The multiple capabilities of HrpJ appear to couple host contact sensing with pilus length control and translocator secretion while also contributing to immunity suppression early in the interaction.     

The Salmonella-containing vacuole: moving with the times

Salmonella pathogenesis is dependent on its ability to invade and replicate within host cells. Following invasion the bacteria remain within a modified phagosome known as the Salmonella-containing vacuole (SCV), within which they will survive and replicate. Invasion and SCV biogenesis are dependent on two Type III secretion systems, T3SS1 and T3SS2, which are used to translocate distinct cohorts of bacterial effector proteins into the host cell. Elucidating the roles of individual effector proteins in SCV biogenesis has proven difficult but several distinct themes are now emerging and it is apparent that SCV biogenesis is an extremely dynamic process involving; extensive membrane remodeling, interactions with the endolysosomal pathway, actin rearrangements and microtubule-based movement and tubule extension.     

Functional relatedness in the Inv/Mxi‐Spa type III secretion system family

Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi‐Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane‐integral pore, and the hydrophilic ‘tip complex’ translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food‐borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi‐Spa family. We used invasion‐deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi‐Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in‐depth survey of the functional interchangeability of Inv/Mxi‐Spa T3SS proteins acting directly at the host‐pathogen interface.     

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.     

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.     

The molecular mechanism of induction of unfolded protein response by Chlamydia

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.     

Characterization of the NleF effector protein from attaching and effacing bacterial pathogens

Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.     

EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function

The enteric pathogens enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Shigella flexneri all translocate at least one effector protein of the EspG protein family into host cells via a type III secretion system (T3SS). The EspG family comprises EspG, EspG2 and VirA. From a Y2H screen, we identified the Golgi matrix protein GM130 as a potential binding partner of EspG. We confirmed EspG:GM130 protein interaction by affinity co-purification. In co-immunoprecipitation experiments EspG was co-precipitated with GM130 while both GM130 and tubulins were co-precipitated with EspG. When expressed ectopically in HeLa cells, the EspG protein family all localized to the Golgi and induced fragmentation of the Golgi apparatus. All EspG family proteins were also able to disrupt protein secretion to a greater extent than the T3SS effector NleA/EspI, which has previously been shown to localize to the Golgi and interact with SEC24 to disrupt COPII vesicle formation. We hypothesize that EspG:GM130 interaction disrupts protein secretion either through direct disruption of GM130 function or through recruitment of other EspG interacting proteins to the Golgi.     

Structural and Biochemical Characterization of SrcA,a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.     

The Type III Secretion Translocation Pore Senses Host Cell Contact

Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip.     

Presence of SopE and mode of infection result in increased Salmonella‐containing vacuole damage and cytosolic release during host cell infection by Salmonella enterica

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that has evolved sophisticated molecular mechanisms to establish an intracellular niche within a specialised vesicular compartment, the Salmonella‐containing vacuole (SCV). The loss of the SCV and release of STM into the cytosol of infected host cells was observed, and a bimodal intracellular lifestyle of STM in the SCV versus life in the cytosol is currently discussed. We set out to investigate the parameters affecting SCV integrity and cytosolic release. A fluorescent protein‐based cytosolic reporter approach was established to quantify, time‐resolved, and on a single cell level, the release of STM into the cytosol of host cells. We observed that the extent of SCV damage and cytosolic release is highly dependent on experimental conditions such as multiplicity of infection, type of host cell line, and STM strain background. Trigger invasion mediated by the Salmonella Pathogenicity Island 1‐encoded type III secretion system (SPI1‐T3SS) and its effector proteins promoted cytosolic release, whereas cytosolic bacteria were rarely observed if entry was mediated by zipper invasion. Presence of SPI1‐T3SS effector SopE was identified as major factor for damage of the SCV in the early phase after STM invasion and sopE‐expressing strains showed higher levels of cytosolic release.