Identification of motifs of Burkholderia pseudomallei BimA required for intracellular motility, actin binding, and actin polymerization

Actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires BimA (Burkholderia intracellular motility A). The mechanism by which BimA mediates actin assembly at the bacterial pole is ill-defined. Toward an understanding of the regions of B. pseudomallei BimA required for intracellular motility and the binding and polymerization of actin, we constructed plasmid-borne bimA variants and glutathione-S-transferase fusion proteins with in-frame deletions of specific motifs. A 13-amino-acid direct repeat and IP₇ proline-rich motif were dispensable for actin binding and assembly in vitro, and expression of the mutated proteins in a B. pseudomallei bimA mutant restored actin-based motility in J774.2 murine macrophage-like cells. However, two WASP homology 2 (WH2) domains were found to be required for actin binding, actin assembly, and plaque formation. A tract of five PDASX direct repeats influenced the polymerization of pyrene-actin monomers in vitro and was required for actin-based motility and intercellular spread, but not actin binding. None of the mutations impaired surface expression or polar targeting of BimA. The number of PDASX repeats varied in natural isolates from two to seven. Such repeats acted additively to promote pyrene-actin polymerization in vitro, with stepwise increases in the rate of polymerization as the number of repeats was increased. No differences in the efficiency of actin tail formation could be discerned between strains expressing BimA variants with two, five, or seven PDASX repeats. The data provide valuable new insights into the role of conserved and variable motifs of BimA in actin-based motility and intercellular spread of B. pseudomallei.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Copper binding in IscA inhibits iron‐sulphur cluster assembly in Escherichia coli

Among the iron‐sulphur cluster assembly proteins encoded by gene cluster iscSUA‐hscBA‐fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron‐sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe‐4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron‐sulphur cluster biogenesis. Here we report that among the iron‐sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA‐mediated [4Fe‐4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe‐4S] clusters in dehydratases, but also block the [4Fe‐4S] cluster assembly in proteins by targeting IscA in cells.     

Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life‐threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin‐binding adaptors p62 and NDP52 and the autophagosome membrane‐associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre‐infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.     

Burkholderia BcpA mediates biofilm formation independently of interbacterial contact‐dependent growth inhibition

Contact‐dependent growth inhibition (CDI) is a phenomenon in which Gram‐negative bacteria use the toxic C‐terminus of a large surface‐exposed exoprotein to inhibit the growth of susceptible bacteria upon cell–cell contact. Little is known about when and where bacteria express the genes encoding CDI system proteins and how these systems contribute to the survival of bacteria in their natural niche. Here we establish that, in addition to mediating interbacterial competition, the Burkholderia thailandensis CDI system exoprotein BcpA is required for biofilm development. We also provide evidence that the catalytic activity of BcpA and extracellular DNA are required for the characteristic biofilm pillars to form. We show using a bcpA–gfp fusion that within the biofilm, expression of the CDI system‐encoding genes is below the limit of detection for the majority of bacteria and only a subset of cells express the genes strongly at any given time. Analysis of a strain constitutively expressing the genes indicates that native expression is critical for biofilm architecture. Although CDI systems have so far only been demonstrated to be involved in interbacterial competition, constitutive production of the system's immunity protein in the entire bacterial population did not alter biofilm formation, indicating a CDI‐independent role for BcpA in this process. We propose, therefore, that bacteria may use CDI proteins in cooperative behaviours, like building biofilm communities, and in competitive behaviours that prevent non‐self bacteria from entering the community.     

A general protein O‐glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence

Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O‐linked protein glycosylation system in B. cenocepacia K56‐2. The PglL_Bc O‐oligosaccharyltransferase (O‐OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the Campylobacter jejuni N‐glycosylation system to a Neisseria meningitides‐derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56‐2, PglL_Bc is responsible for the glycosylation of 23 proteins involved in diverse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc‐HexNAc‐Hex, which is unrelated to the O‐antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first example of post‐translational modification in Bcc with implications for pathogenesis.     

Single‐molecule atomic force microscopy unravels the binding mechanism of a Burkholderia cenocepacia trimeric autotransporter adhesin

Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram‐negative bacteria. Prominent examples of TAAs are found in Burkholderia cepacia complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that Burkholderia cenocepacia TAAs mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single‐molecule atomic force microscopy to unravel the binding mechanism of BCAM0224, a prototype TAA from B. cenocepacia K56‐2. We show that the adhesin forms homophilic trans‐interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that BCAM0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that BCAM0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.     

Pathogen–host reorganization during Chlamydia invasion revealed by cryo‐electron tomography

Invasion of host cells is a key early event during bacterial infection, but the underlying pathogen–host interactions are yet to be fully visualized in three‐dimensional detail. We have captured snapshots of the early stages of bacterial‐mediated endocytosis in situ by exploiting the small size of chlamydial elementary bodies (EBs) for whole‐cell cryo‐electron tomography. Chlamydiae are obligate intracellular bacteria that infect eukaryotic cells and cause sexually transmitted infections and trachoma, the leading cause of preventable blindness. We demonstrate that Chlamydia trachomatis LGV2 EBs are intrinsically polarized. One pole is characterized by a tubular inner membrane invagination, while the other exhibits asymmetric periplasmic expansion to accommodate an array of type III secretion systems (T3SSs). Strikingly, EBs orient with their T3SS‐containing pole facing target cells, enabling the T3SSs to directly contact the cellular plasma membrane. This contact induces enveloping macropinosomes, actin‐rich filopodia and phagocytic cups to zipper tightly around the internalizing bacteria. Once encapsulated into tight early vacuoles, EB polarity and the T3SSs are lost. Our findings reveal previously undescribed structural transitions in both pathogen and host during the initial steps of chlamydial invasion.     

A β1‐6/β1‐3 galactosidase from Bifidobacterium animalis subsp. lactis Bl‐04 gives insight into sub‐specificities of β‐galactoside catabolism within Bifidobacterium

The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host‐derived β‐galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β‐galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl‐04 (BlGal42A). BlGal42A displays a preference for undecorated β1‐6 and β1‐3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N‐acetyl substituted β1‐3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite + 1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite ? 1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1‐6 and β1‐3 galactosides likely reflects structural differences between these substrates and β1‐4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub‐specificities that support metabolic specialization in the gut niche.     

Myosin‐X facilitates Shigella‐induced membrane protrusions and cell‐to‐cell spread

The intracellular pathogen Shigella flexneri forms membrane protrusions to spread from cell to cell. As protrusions form, myosin‐X (Myo10) localizes to Shigella. Electron micrographs of immunogold‐labelled Shigella‐infected HeLa cells reveal that Myo10 concentrates at the bases and along the sides of bacteria within membrane protrusions. Time‐lapse video microscopy shows that a full‐length Myo10 GFP‐construct cycles along the sides of Shigella within the membrane protrusions as these structures progressively lengthen. RNAi knock‐down of Myo10 is associated with shorter protrusions with thicker stalks, and causes a >80% decrease in confluent cell plaque formation. Myo10 also concentrates in membrane protrusions formed by another intracellular bacteria, Listeria, and knock‐down of Myo10 also impairs Listeria plaque formation. In Cos7 cells (contain low concentrations of Myo10), the expression of full‐length Myo10 nearly doubles Shigella‐induced protrusion length, and lengthening requires the head domain, as well as the tail‐PH domain, but not the FERM domain. The GFP‐Myo10‐HMM domain localizes to the sides of Shigella within membrane protrusions and the GFP‐Myo10‐PH domain localizes to host cell membranes. We conclude thatMyo10 generates the force to enhance bacterial‐induced protrusions by binding its head region to actin filaments and its PH tail domain to the peripheral membrane.     

Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid‐producing bacteria

Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C₃₅ extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild‐type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid‐producing bacteria.     

Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis

Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly “highly resistant” to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis.     

Activation and polar sequestration of PopA,a c‐di‐GMP effector protein involved in Caulobacter crescentus cell cycle control

When Caulobacter crescentus enters S‐phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co‐option as c‐di‐GMP effector protein. While the C‐terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N‐terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N‐terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c‐di‐GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S‐phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.     

A bacterial effector targets host DH‐PH domain RhoGEFs and antagonizes macrophage phagocytosis

Bacterial pathogens often harbour a type III secretion system (TTSS) that injects effector proteins into eukaryotic cells to manipulate host processes and cause diseases. Identification of host targets of bacterial effectors and revealing their mechanism of actions are crucial for understating bacterial virulence. We show that EspH, a type III effector conserved in enteric bacterial pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli and Citrobacter rodentium, markedly disrupts actin cytoskeleton structure and induces cell rounding up when ectopically expressed or delivered into HeLa cells by the bacterial TTSS. EspH inactivates host Rho GTPase signalling pathway at the level of RhoGEF. EspH directly binds the DH‐PH domain in multiple RhoGEFs, which prevents their binding to Rho and thereby inhibits nucleotide exchange‐mediated Rho activation. Consistently, infection of mouse macrophages with EPEC harbouring EspH attenuates phagocytosis of the bacteria as well as FcγR‐mediated phagocytosis. EspH represents the first example of targeting RhoGEFs by bacterial effectors, and our results also reveal an unprecedented mechanism used by enteric pathogens to counteract the host defence system.     

Actin‐based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin‐based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin‐based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time‐lapse microscopy using green fluorescent protein‐LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin‐based motility moved away from LC3‐positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin‐based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol.     

The role of FlhF and HubP as polar landmark proteins in Shewanella putrefaciens CN‐32

Spatiotemporal regulation of cell polarity plays a role in many fundamental processes in bacteria and often relies on ‘landmark’ proteins which recruit the corresponding clients to their designated position. Here, we explored the localization of two multi‐protein complexes, the polar flagellar motor and the chemotaxis array, in Shewanella putrefaciens CN‐32. We demonstrate that polar positioning of the flagellar system, but not of the chemotaxis system, depends on the GTPase FlhF. In contrast, the chemotaxis array is recruited by a transmembrane protein which we identified as the functional ortholog of Vibrio cholerae HubP. Mediated by its periplasmic N‐terminal LysM domain, SpHubP exhibits an FlhF‐independent localization pattern during cell cycle similar to its Vibrio counterpart and also has a role in proper chromosome segregation. In addition, while not affecting flagellar positioning, SpHubP is crucial for normal flagellar function and is involved in type IV pili‐mediated twitching motility. We hypothesize that a group of HubP/FimV homologs, characterized by a rather conserved N‐terminal periplasmic section required for polar targeting and a highly variable acidic cytoplasmic part, primarily mediating recruitment of client proteins, serves as polar markers in various bacterial species with respect to different cellular functions.     

PfeT,a P1B4‐type ATPase,effluxes ferrous iron and protects Bacillus subtilis against iron intoxication

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P_1B4‐type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild‐type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini‐ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P_1B4‐type ATPases previously implicated in bacterial pathogenesis.