RACK1 promotes Shigella flexneri actin-mediated invasion, motility, and cell-to-cell spreading.

Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.

Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements.

Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.

Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

The Functional Differences between the GroEL Chaperonin of Escherichia coli and the HtpB Chaperonin of Legionella pneumophila Can Be Mapped to Specific Amino Acid Residues.

Group I chaperonins are a highly conserved family of essential proteins that self-assemble into molecular nanoboxes that mediate the folding of cytoplasmic proteins in bacteria and organelles. GroEL, the chaperonin of Escherichia coli, is the archetype of the family. Protein folding-independent functions have been described for numerous chaperonins, including HtpB, the chaperonin of the bacterial pathogen Legionella pneumophila. Several protein folding-independent functions attributed to HtpB are not shared by GroEL, suggesting that differences in the amino acid (aa) sequence between these two proteins could correlate with functional differences. GroEL and HtpB differ in 137 scattered aa positions. Using the Evolutionary Trace (ET) bioinformatics method, site-directed mutagenesis, and a functional reporter test based upon a yeast-two-hybrid interaction with the eukaryotic protein ECM29, it was determined that out of those 137 aa, ten (M68, M212, S236, K298, N507 and the cluster AEHKD in positions 471-475) were involved in the interaction of HtpB with ECM29. GroEL was completely unable to interact with ECM29, but when GroEL was modified at those 10 aa positions, to display the HtpB aa, it acquired a weak ability to interact with ECM29. This constitutes proof of concept that the unique functional abilities of HtpB can be mapped to specific aa positions.

Disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate lyase enhances pathogen eradication by antibiotics.

BACKGROUND: Pseudomonas aeruginosa forms antibiotic-resistant biofilms that are responsible for the treatment failure or relapses of the bacterial infections in the lungs of patients with cystic fibrosis (CF). The alginate lyases that target extracellular polysaccharide alginate of P. aeruginosa biofilms are promising therapeutic candidates for treatment of P. aeruginosa biofilm infections. METHODS: Immunofluorescent staining and thin layer chromatography were used to demonstrate the alginolytic activity of the alginate lyase enzyme (AlyP1400) purified from a marine Pseudoalteromonas bacterium. Anti-biofilm activities of AlyP1400 were tested alone or in combination with antibiotics on the biofilms of a mucoid Pseudomonas aeruginosa clinical isolate CF27 that were cultivated in 96-well plates and a flow cell. RESULTS: We showed that AlyP1400 facilitated antibiotic activities to eliminate CF27 biofilms. The combination of AlyP1400 with antibiotics reduced the biofilm biomass and boosted bactericidal activity of antibiotics. Importantly, we demonstrated that the enzymatic activity of AlyP1400 was required for its biofilm disruption activity and its synergy with antibiotics to eradicate biofilm cells. CONCLUSION: This work shed new light on the potential mechanisms of the therapeutic activity for the combinational use of alginate lyase and antibiotics to treat P. aeruginosa infections in CF lungs or other P. aeruginosa biofilm-related infections.

Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

Marine Bacteria, A Source for Alginolytic Enzyme to Disrupt Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.

Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria.

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.

Taxonomic differences of gut microbiomes drive cellulolytic enzymatic potential within hind-gut fermenting mammals.

Host diet influences the diversity and metabolic activities of the gut microbiome. Previous studies have shown that the gut microbiome provides a wide array of enzymes that enable processing of diverse dietary components. Because the primary diet of the porcupine, Erethizon dorsatum, is lignified plant material, we reasoned that the porcupine microbiome would be replete with enzymes required to degrade lignocellulose. Here, we report on the bacterial composition in the porcupine microbiome using 16S rRNA sequencing and bioinformatics analysis. We extended this analysis to the microbiomes of 20 additional mammals located in Shubenacadie Wildlife Park (Nova Scotia, Canada), enabling the comparison of bacterial diversity amongst three mammalian taxonomic orders (Rodentia, Carnivora, and Artiodactyla). 16S rRNA sequencing was validated using metagenomic shotgun sequencing on selected herbivores (porcupine, beaver) and carnivores (coyote, Arctic wolf). In the microbiome, functionality is more conserved than bacterial composition, thus we mined microbiome data sets to identify conserved microbial functions across species in each order. We measured the relative gene abundances for cellobiose phosphorylase, endoglucanase, and beta-glucosidase to evaluate the cellulose-degrading potential of select mammals. The porcupine and beaver had higher proportions of genes encoding cellulose-degrading enzymes than the Artic wolf and coyote. These findings provide further evidence that gut microbiome diversity and metabolic capacity are influenced by host diet.

Innate Recognition of Intracellular Bacterial Growth Is Driven by the TIFA-Dependent Cytosolic Surveillance Pathway.

Intestinal epithelial cells (IECs) act as sentinels for incoming pathogens. Cytosol-invasive bacteria, such as Shigella flexneri, trigger a robust pro-inflammatory nuclear factor κB (NF-κB) response from IECs that is believed to depend entirely on the peptidoglycan sensor NOD1. We found that, during Shigella infection, the TRAF-interacting forkhead-associated protein A (TIFA)-dependent cytosolic surveillance pathway, which senses the bacterial metabolite heptose-1,7-bisphosphate (HBP), functions after NOD1 to detect bacteria replicating free in the host cytosol. Whereas NOD1 mediated a transient burst of NF-κB activation during bacterial entry, TIFA sensed HBP released during bacterial replication, assembling into large signaling complexes to drive a dynamic inflammatory response that reflected the rate of intracellular bacterial proliferation. Strikingly, IECs lacking TIFA were unable to discriminate between proliferating and stagnant intracellular bacteria, despite the NOD1/2 pathways being intact. Our results define TIFA as a rheostat for intracellular bacterial replication, escalating the immune response to invasive Gram-negative bacteria that exploit the host cytosol for growth.

Shigella infection interferes with SUMOylation and increases PML-NB number.

Shigellosis is a severe diarrheal disease that affects hundreds of thousands of individuals resulting in significant morbidity and mortality worldwide. Shigellosis is caused by Shigella spp., a gram-negative bacterium that uses a Type 3 Secretion System (T3SS) to deliver effector proteins into the cytosol of infected human cells. Shigella infection triggers multiple signaling programs that result in a robust host transcriptional response that includes the induction of multiple proinflammatory cytokines. PML nuclear bodies (PML-NBs) are dynamic subnuclear structures that coordinate immune signaling programs and have a demonstrated role in controlling viral infection. We show that PML-NB number increases upon Shigella infection. We examined the effects of Shigella infection on SUMOylation and found that upon Shigella infection the localization of SUMOylated proteins is altered and the level of SUMOylated proteins decreases. Although Shigella infection does not alter the abundance of SUMO activating enzymes SAE1 or SAE2, it dramatically decreases the level of the SUMO conjugating enzyme Ubc9. All Shigella-induced alterations to the SUMOylation system are dependent upon a T3SS. Thus, we demonstrate that Shigella uses one or more T3SS effectors to influence both PML-NB number and the SUMOylation machinery in human cells.

Visualization of the type III secretion sorting platform of Shigella flexneri.

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.

The bacterial pathogen-ubiquitin interface: lessons learned from Shigella.

Shigella species are the aetiological agents of shigellosis, a severe diarrhoeal disease that is a significant cause of morbidity and mortality worldwide. Shigellosis causes massive colonic destruction, high fever and bloody diarrhoea. Shigella pathogenesis is tightly linked to the ability of the bacterium to invade and replicate intracellularly within the colonic epithelium. Shigella uses a type 3 secretion system to deliver its effector proteins into the cytosol of infected cells. Among the repertoire of Shigella effectors, many are known to target components of the actin cytoskeleton to promote bacterial entry. An emerging alternate theme for effector function is the targeting of the host ubiquitin system. Ubiquitination is a post-translational modification restricted to eukaryotes and is involved in many essential host processes. By virtue of sheer number of ubiquitin-modulating effector proteins, it is clear that Shigella has invested heavily into subversion of the ubiquitin system. Understanding these host-pathogen interactions will inform us about the strategies used by successful pathogens and may also provide avenues for novel antimicrobial strategies.

A Shigella flexneri virulence plasmid encoded factor controls production of outer membrane vesicles.

Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA.

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Structure of an SspH1-PKN1 complex reveals the basis for host substrate recognition and mechanism of activation for a bacterial E3 ubiquitin ligase.

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.

Genome sequence of Shigella flexneri serotype 5a strain M90T Sm.

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).

Conserved structural mechanisms for autoinhibition in IpaH ubiquitin ligases.

The IpaH family of novel E3 ligase (NEL) enzymes occur in a variety of pathogenic and commensal bacteria that interact with eukaryotic hosts. We demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes autoinhibit the enzymatic activity of the adjacent catalytic novel E3 ligase domain by two distinct but conserved structural mechanisms. Autoinhibition is required for the in vivo biological activity of two IpaH enzymes in a eukaryotic model system. Autoinhibition was retro-engineered into a constitutively active IpaH enzyme from Yersinia pestis by introduction of single site substitutions, thereby demonstrating the conservation of autoregulatory infrastructure across the IpaH enzyme family.