Joe Pogliano

Bacteriophage adhering to mucus provide a non–host-derived immunity

Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phage-to-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non–host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.

ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase

Broad‐host‐range plasmid RK2 encodes a post‐segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication‐proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase‐ and gyrase‐dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase–DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.

A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation

At the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4‐64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild‐type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site. Using time‐lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation. Septal dissolution was dependent on σE activity and was partially inhibited in mutants lacking the σE‐controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan. Our results support a model in which σE inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before σE activation.

SecD and SecF facilitate protein export in Escherichia coli.

We show here that the rate of protein translocation in the bacterium Escherichia coli depends on the levels of the SecD and SecF proteins in the cell. Overexpression of SecD and SecF stimulates translocation in wild type cells and improves export of proteins with mutant signal sequences. Depletion of SecD and SecF from the cell greatly reduces but does not abolish protein translocation. A secDF::kan null mutant deleted for the genes encoding both proteins is cold‐sensitive for growth and protein export, has a severe export defect at 37 degrees C and is barely viable. The phenotypes of a secD null mutant and a secF null mutant are identical to the secDF::kan double null mutant. These results partially resolve the conflict between genetic studies and results from in vitro translocation systems which do not require SecD and SecF for activity, affirm the importance of these proteins to the export process, and suggest that SecD and SecF function together to stimulate protein export in a role fundamentally different from other Sec proteins. Our results provide additional support for the notion that an early step in protein export is cold‐sensitive.

Daptomycin-Mediated Reorganization of Membrane Architecture Causes Mislocalization of Essential Cell Division Proteins

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

Multicopy plasmids are clustered and localized in Escherichia coli

We localized the multicopy plasmid RK2 in Escherichia coli and found that the number of fluorescent foci observed in each cell was substantially less than the copy number of the plasmid, suggesting that many copies of RK2 are grouped into a few multiplasmid clusters. In minimal glucose media, the majority of cells had one or two foci, with a single focus localized near midcell, and two foci near the 1/4 and 3/4 cell positions. The number of foci per cell increased with cell length and with growth rate, and decreased upon entering stationary phase, suggesting a coordination of RK2 replication or segregation with the bacterial cell cycle. Time-lapse microscopy demonstrated that partitioning of RK2 foci is achieved by the splitting of a single focus into two or three smaller foci, which are capable of separating with rapid kinetics. A derivative of the high-copy-number plasmid pUC19 containing the lacO array was also localized by tagging with GFP-LacI. Whereas many of the cells contained numerous, randomly diffusing foci, most cells exhibited one or two plasmid clusters located at midcell or the cell quarter positions. Our results suggest a model in which multicopy plasmids are not always randomly diffusing throughout the cell as previously thought, but can be replicated and partitioned in clusters targeted to specific locations.

Ampicillin Enhances Daptomycin- and Cationic Host Defense Peptide-Mediated Killing of Ampicillin- and Vancomycin-Resistant Enterococcus faecium

ABSTRACT We studied an ampicillin- and vancomycin-resistant Enterococcus faecium (VRE) isolate from a patient with endocarditis and bacteremia refractory to treatment with daptomycin (6 mg/kg of body weight) plus linezolid. Blood cultures cleared within 24 h of changing therapy to daptomycin (12 mg/kg) plus ampicillin. We examined the effects of ampicillin on daptomycin-induced growth inhibition and killing, surface charge, and susceptibility to several prototypical host defense cationic antimicrobial peptides. MICs and time-kill curves with daptomycin were assessed in the presence and absence of ampicillin. The impact of ampicillin on surface charge was assessed by flow cytometry and a poly-l-lysine binding assay. The effects of ampicillin preexposures upon VRE killing by five distinct cationic peptides of different structure, charge, origin, and mechanism of action were analyzed using the epidermal cathelicidin LL-37, thrombin-induced platelet microbicidal proteins (tPMPs), and a synthetic congener modeled after tPMP microbicidal domains (RP-1), human neutrophil peptide-1 (hNP-1), and polymyxin B (bacteria derived). Fluoroscein-Bodipy-labeled daptomycin was used to evaluate daptomycin binding to VRE membranes in the presence or absence of ampicillin. In media containing ampicillin (25 to 100 mg/liter), daptomycin MICs decreased from 1.0 to 0.38 mg/liter. Based on time-kill analysis and an in vitro pharmacodynamic model, ampicillin enhanced daptomycin activity against the study VRE from a bacteriostatic to a bactericidal profile. VRE grown in ampicillin (25 to 150 mg/liter) demonstrated an incremental reduction in its relative net positive surface charge. When grown in the presence (versus absence) of ampicillin (25 and 100 mg/liter), the VRE strain (i) was more susceptible to killing by LL-37, tPMPs, hNP-1, and RP-1 but not to polymyxin B and (ii) exhibited greater binding to Bodipy-labeled daptomycin. We conclude that ampicillin induces reductions in net positive bacterial surface charge of VRE, correlating with enhanced bactericidal effects of cationic calcium-daptomycin and a diverse range of other cationic peptides in vitro. While the mechanism(s) of such β-lactam-mediated shifts in surface charge remains to be defined, these finding suggest a potential for β-lactam-mediated enhancement of activity of both daptomycin and innate host defense peptides against antibiotic-resistant bacteria.

Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin‐like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.

DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development.

We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32‐derivative) during growth and sporulation. A 3‐kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA‐GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t1/2<∼45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two‐fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.