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Background Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. Results We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells. Conclusions Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.

Recalcitrant chronic infections of implanted medical devices are often linked to the presence of biofilms. The prevention and treatment of medical device-associated infections is a major source of antibiotic use and driver of antimicrobial resistance globally. Lowering the incidence of infection in patients that receive implanted medical devices could therefore significantly improve antibiotic stewardship and reduce patient morbidity. Here we determined if modifying the design of an implantable medical device to reduce bacterial attachment, impacted the incidence of device-associated infections in clinical practice. Since the 1980s cochlear implants have provided long-term treatment of sensorineural hearing deficiency in hundreds of thousands of patients world-wide. Nonetheless, a relatively small number of devices are surgically explanted each year due to unresolvable infections. Features associated with the accumulation of bacteria on the Cochlear™ Nucleus® CI24RE™ model of cochlear implant devices were identified using both in vitro bacterial attachment assays and examination of explanted devices. Macro-scale design modifications that reduced bacterial attachment in vitro were incorporated into the design of the CI500™ and Profile™ series of Nucleus implant. Analyses of mandatory post-market vigilance data of 198,757 CI24RE and 123,084 CI500/Profile series implantation surgeries revealed that these design modifications correlated with significantly reduced infection rates. This study demonstrates that a design-centric approach aimed at mitigating bacterial attachment was a simple, and effective means of reducing infections associated with Cochlear Nucleus devices. This approach is likely to be applicable to improving the designs of other implantable medical devices to reduce device-associated infections.

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

This work highlights the multifunctional role of the ISG viperin and its interaction with the peroxisomal protein Pex19 to modulate peroxisomal-dependent innate signaling that ultimately restricts viral infection., Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-β. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa .

Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate twitching motility via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has an MCP PilJ and two CheW adapter proteins, PilI and ChpC, that likely interact with the histidine kinase ChpA to feed environmental signals into the system. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3′−5′-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Furthermore, we show that protease activity is required for the twitching motility response of P. aeruginosa to environmental signals. Based upon our data we propose a model whereby ChpC feeds these environmental signals into the Chp system, potentially via PilJ or another MCP, to control twitching motility. PilJ and PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa .

Chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. Bacterial biofilms cause most chronic wound infections and are notoriously resistant to antibiotic treatments. The need for new approaches to combat polymicrobial biofilms in chronic wounds combined with the growing antimicrobial resistance crisis means that honey is being revisited as a treatment option due to its broad-spectrum antimicrobial activity and low propensity for bacterial resistance. We assessed four well-characterised New Zealand honeys, quantified for their key antibacterial components, methylglyoxal, hydrogen peroxide and sugar, for their capacity to prevent and eradicate biofilms produced by the common wound pathogen Pseudomonas aeruginosa. We demonstrate that: (1) honey used at substantially lower concentrations compared to those found in honey-based wound dressings inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms; (2) the anti-biofilm effect of honey was largely driven by its sugar component; (3) cells recovered from biofilms treated with sub-inhibitory honey concentrations had slightly increased tolerance to honey; and (4) honey used at clinically obtainable concentrations completely eradicated established P. aeruginosa biofilms. These results, together with their broad antimicrobial spectrum, demonstrate that manuka honey-based wound dressings are a promising treatment for infected chronic wounds, including those with P. aeruginosa biofilms.

Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate twitching motility via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has an MCP PilJ and two CheW adapter proteins, PilI and ChpC, that likely interact with the histidine kinase ChpA to feed environmental signals into the system. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3′−5′-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Furthermore, we show that protease activity is required for the twitching motility response of P. aeruginosa to environmental signals. Based upon our data we propose a model whereby ChpC feeds these environmental signals into the Chp system, potentially via PilJ or another MCP, to control twitching motility. PilJ and PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa .

Background: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. Multiple different functions and cellular locations have been attributed to PGRMC1 in a variety of contexts, however the mechanisms underlying PGRMC1 biology remain obscure. The protein contains several phosphorylated residues including tyrosines which were acquired in animal evolution prior to bilateral symmetry, and could be involved with animal cell differentiation mechanisms. Results: Here we demonstrate that mutagenic manipulation of PGRMC1 phosphorylation status in MIA PaCa-2 pancreatic cells exerts broad pleiotropic effects, influencing cell plasticity and tumorigenicity, as assayed by cell biological and proteomics measurements. Relative to parental cells over-expressing hemagglutinin-tagged wild-type PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with Rho-kinase inhibitor-sensitive changes including altered cell shape, motility, increased PI3K/Akt activity, and fragmented mitochondrial morphology. An S57A/Y180F/S181A triple mutant reduced PI3K/Akt activity, indicating involvement of Y180 in PI3K/Akt induction. Both triple mutant and Y180F single mutant cells exhibited attenuated mouse xenograft tumor growth. Conclusions: Phosphorylation status of the PGRMC1 tyrosine 180 regulatory motif exerts dramatic influence over cancer cell biology, including Warburg effect-like glucose metabolism. In accompanying papers we show that: 1) the cells examined here exhibit dramatically altered metabolism and epigenetic status, with the triple mutant inducing hypermethylation similar to that of embryonic stem cells, and that 2) Y180 was acquired in evolution concurrently with appearance of the organizer that induces animal gastrulation. Taken together, these results indicate that the undescribed mechanisms regulating PGRMC1 phosphorylation may be of great disease relevance and merit urgent investigation.

Pandoraea species have been isolated from diverse environmental samples and are emerging important respiratory pathogens, particularly in people with cystic fibrosis (CF). In the present study, two bacterial isolates initially recovered from consecutive sputum samples collected from a CF patient and identified as Pandoraea pnomenusa underwent a polyphasic taxonomic analysis. The isolates were found to be Gram-negative, facultative anaerobic motile bacilli and subsequently designated as strains 6399T (=LMG29626T=DSM103228T) and 7641 (=LMG29627=DSM103229), respectively. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences revealed that 6399T and 7641 formed a distinct phylogenetic lineage within the genus Pandoraea. Genome sequence comparison analysis indicated that strains 6399T and 7641 are clonal and share 100 % similarity, however, similarity to other type strains (ANIb 73.2-88.8 %, ANIm 83.5-89.9 % and OrthoANI 83.2-89.3 %) indicates that 6399T and 7641 do not belong to any of the reported type species. The major cellular fatty acids of 6399T were C16 : 0 (32.1 %) C17 : 0cyclo (18.7 %) and C18 : 1ω7c (14.5 %), while Q-8 was the only respiratory quinone detected. The major polar lipids identified were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of 6399T was 62.9 (mol%). Strain 6399T can be differentiated from other members of Pandoraea by the absence of C19 : 0ω8c cyclo and by the presence of C17 : 0ω8c cyclo. Together our data show that the bacterial strains 6399T and 7641 represent a novel species of the genus Pandoraea, for which the name Pandoraea fibrosis sp. nov. is proposed (type strain 6399T).

Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+)-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+)-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+)-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.

Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.

Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin β1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin β1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin β1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.

Mycoplasma hyopneumoniae is an economically devastating, globally disseminated pathogen that can maintain a chronic infectious state within its host, swine. Here, we depict the events underpinning M. hyopneumoniae biofilm formation on an abiotic surface and demonstrate for the first time, biofilms forming on porcine epithelial cell monolayers and in the lungs of pigs, experimentally infected with M. hyopneumoniae. Nuclease treatment prevents biofilms forming on glass but not on porcine epithelial cells indicating that extracellular DNA (eDNA), which localises at the base of biofilms, is critical in the formation of these structures on abiotic surfaces. Subpopulations of M. hyopneumoniae cells, denoted by their ability to take up the dye TOTO-1 and release eDNA, were identified. A visually distinct sub-population of pleomorphic cells, that we refer to here as large cell variants (LCVs), rapidly transition from phase dark to translucent “ghost” cells. The translucent cells accumulate the membrane-impermeable dye TOTO-1, forming readily discernible membrane breaches immediately prior to lysis and the possible release of eDNA and other intracellular content (public goods) into the extracellular environment. Our novel observations expand knowledge of the lifestyles adopted by this wall-less, genome-reduced pathogen and provide further insights to its survival within farm environments and swine.

© 2018 Liu, Cokcetin, Lu, Turnbull, Carter, Whitchurch and Harry. Chronic wound infections are a major burden to both society and the health care industry. Bacterial biofilms are the major cause of chronic wound infections and are notoriously recalcitrant to treatments with antibiotics, making them difficult to eradicate. Thus, new approaches are required to combat biofilms in chronic wounds. One possible approach is to use drug combination therapies. Manuka honey has potent broad-spectrum antibacterial activity and has previously shown synergistic activity in combination with antibiotics against common wound pathogens, including Staphylococcus aureus. In addition, manuka honey exhibits anti-biofilm activity, thereby warranting the investigation of its potential as a combination therapy with antibiotics for the topical treatment of biofilm-related infections. Here we report the first use of MacSynergy II to investigate the response of established S. aureus (strain NCTC 8325) biofilms to treatment by combinations of Medihoney (medical grade manuka honey) and conventional antibiotics that are used for preventing or treating infections: rifampicin, oxacillin, fusidic acid, clindamycin, and gentamicin. Using checkerboard microdilution assays, viability assays and MacSynergy II analysis we show that the Medihoney-rifampicin combination was more effective than combinations using the other antibiotics against established staphylococcal biofilms. Medihoney and rifampicin were strongly synergistic in their ability to reduce both biofilm biomass and the viability of embedded S. aureus cells at a level that is likely to be significant in vivo. Other combinations of Medihoney and antibiotic produced an interesting array of effects: Medihoney-fusidic acid treatment showed minor synergistic activity, and Medihoney-clindamycin, -gentamicin, and -oxacillin combinations showed overall antagonistic effects when the honey was used at sub-inhibitory concentration, due to enhanced biofilm formation at these concentrations which could not be counteracted by the antibiotics. However, these combinations were not antagonistic when honey was used at the inhibitory concentration. Confocal scanning laser microscopy confirmed that different honey-antibiotic combination treatments could eradicate biofilms. Our results suggest that honey has potential as an adjunct treatment with rifampicin for chronic wounds infected with staphylococcal biofilms. We also show that MacSynergy II allows a comprehensive examination of the synergistic effects of honey-antibiotic combinations, and can help to identify doses for clinical use.

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems., The three-dimensional architecture of genome-reduced bacteria is poorly understood. Here the authors combine Hi-C with super-resolution microscopy in Mycoplasma pneumoniae and provide evidence of how supercoiling and local organization influences gene regulation.

© 2017 Gloag, Elbadawi, Zachreson, Aharonovich, Toth, Charles, Turnbull and Whitchurch. Twitching motility is a mode of surface translocation that is mediated by the extension and retraction of type IV pili and which, depending on the conditions, enables migration of individual cells or can manifest as a complex multicellular collective behavior that leads to biofilm expansion. When twitching motility occurs at the interface of an abiotic surface and solidified nutrient media, it can lead to the emergence of extensive self-organized patterns of interconnected trails that form as a consequence of the actively migrating bacteria forging a furrow network in the substratum beneath the expanding biofilm. These furrows appear to direct bacterial movements much in the same way that roads and footpaths coordinate motor vehicle and human pedestrian traffic. Self-organizing systems such as these can be accounted for by the concept of stigmergy which describes self-organization that emerges through indirect communication via persistent signals within the environment. Many bacterial communities are able to actively migrate across solid and semi-solid surfaces through complex multicellular collective behaviors such as twitching motility and flagella-mediated swarming motility. Here, we have examined the potential of exploiting the stigmergic behavior of furrow-mediated trail following as a means of controlling bacterial biofilm expansion along abiotic surfaces. We found that incorporation of a series of parallel micro-fabricated furrows significantly impeded active biofilm expansion by Pseudomonas aeruginosa and Proteus vulgaris. We observed that in both cases bacterial movements tended to be directed along the furrows. We also observed that narrow furrows were most effective at disrupting biofilm expansion as they impeded the ability of cells to self-organize into multicellular assemblies required for escape from the furrows and migration into new territory. Our results suggest that the implementation of micro-fabricated furrows that exploit stigmergy may be a novel approach to impeding active biofilm expansion across abiotic surfaces such as those used in medical and industrial settings.

© Springer Science+Business Media New York 2017. Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.

[Summary]: Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.

Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethylenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix strength of biofilms of several bacterial species as well as to have bactericidal activity against various pathogens. EDTA exerts its antimicrobial activity by chelating divalent cations necessary for growth and membrane stability and by destabilizing the matrix thus enhancing the detachment of bacterial cells from the biofilm. In this study, we have explored the role of divalent cations in NTHi biofilm development and stability. We have utilized in vitro static and continuous flow models of biofilm development by NTHi to demonstrate that magnesium cations enhance biofilm formation by NTHi. We found that the divalent cation chelator EDTA is effective at both preventing NTHi biofilm formation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections.

The Gram-negative human pathogen Pseudomonas aeruginosa tolerates high concentrations of β-lactam antibiotics. Despite inhibiting the growth of the organism, these cell wall-targeting drugs exhibit remarkably little bactericidal activity. However, the mechanisms underlying β-lactam tolerance are currently unclear. Here, we show that P. aeruginosa undergoes a rapid en masse transition from normal rod-shaped cells to viable cell wall-defective spherical cells when treated with β-lactams from the widely used carbapenem and penicillin classes. When the antibiotic is removed, the entire population of spherical cells quickly converts back to the normal bacillary form. Our results demonstrate that these rapid population-wide cell morphotype transitions function as a strategy to survive antibiotic exposure. Taking advantage of these findings, we have developed a novel approach to efficiently kill P. aeruginosa by using carbapenem treatment to induce en masse transition to the spherical cell morphotype and then exploiting the relative fragility and sensitivity of these cells to killing by antimicrobial peptides (AMPs) that are relatively inactive against P. aeruginosa bacillary cells. This approach could broaden the repertoire of antimicrobial compounds used to treat P. aeruginosa and serve as a basis for developing new therapeutic agents to combat bacterial infections.

Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.

We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

The study of host-pathogen interactions over past decades has benefited from advances in microscopy and fluorescent imaging techniques. A particularly powerful model in this field is vaccinia virus (VACV), which due to its amenability to genetic manipulation has been a productive model in advancing the understanding of the transport of subcellular cargoes. Conventional light microscopy imposes an upper limit of resolution of ∼250. nm, hence knowledge of events occurring at the sub-viral resolution is based predominantly on studies utilising electron microscopy. The development of super-resolution light microscopy presents the opportunity to bridge the gap between these two technologies. This report describes the analysis of VACV replication using fluorescent recombinant viruses, achieving sub-viral resolution with three-dimensional structured illumination microscopy. This is the first report of successfully resolving poxvirus particle morphologies at the scale of single virus particles using light microscopy. © 2012 Elsevier B.V.

Surface translocation by the soil bacterium Myxococcus xanthus is a complex multicellular phenomenon that entails two motility systems. However, the mechanisms by which the activities of individual cells are coordinated to manifest this collective behaviour are currently unclear. Here we have developed a novel assay that enables detailed microscopic examination of M. xanthus motility at the interstitial interface between solidified nutrient medium and a glass coverslip. Under these conditions, M. xanthus motility is characterised by extensive micro-morphological patterning that is considerably more elaborate than occurs at an air-surface interface. We have found that during motility on solidified nutrient medium, M. xanthus forges an interconnected furrow network that is lined with an extracellular matrix comprised of exopolysaccharides, extracellular lipids, membrane vesicles and an unidentified slime. Our observations have revealed that M. xanthus motility on solidified nutrient medium is a stigmergic phenomenon in which multi-cellular collective behaviours are co-ordinated through trail-following that is guided by physical furrows and extracellular matrix materials.

Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.

Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant 2-fold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a 2-fold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin. © 2012 Institut Pasteur.