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.

Combining Whole-Genome Sequencing and Multimodel Phenotyping To Identify Genetic Predictors of Salmonella Virulence.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.

Subcellular Location of Piscirickettsia salmonis Heat Shock Protein 60 (Hsp60) Chaperone by Using Immunogold Labeling and Proteomic Analysis.

Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by the T4SS, T3SS, and T6SS, with negative results. These data support the hypothesis that small amounts of Hsp60 must reach the bacterial cell surface in a manner probably not mediated by currently characterized secretion systems, and that they remain biologically active during P. salmonis infection, possibly mediating adherence and (or) invasion.

Potential Ad Hoc Markers of Persistence and Virulence in Canadian Listeria monocytogenes Food and Clinical Isolates.

The Listeria monocytogenes gene inlA, encoding a surface virulence protein, was examined for the presence of premature stop codon (PMSC) mutations in 82 isolates obtained by the Canadian Food Inspection Agency (CFIA) from foods and food contact surfaces. These mutations were coanalyzed for the presence of stress survival islet 1 (SSI-1) and for the abilities of the isolates to invade Caco-2 intestinal epithelial cells and form biofilms on polystyrene. PMSC mutations were present in one-third of the isolates (predominantly those of serogroup 1/2a), and their presence was correlated with a noninvasive phenotype. The presence of SSI-1 and the ability to form biofilms were also linked to the 1/2a serogroup. Serogroup 4b isolates lacked inlA PMSC mutations and were invasive, but neither formed biofilms nor carried SSI-1. To expand upon these experimental findings, an in silico analysis was performed on L. monocytogenes genomes from Canadian databases of 278 food isolates and 607 clinical isolates. The prevalence of inlA PMSC mutations in genomes of food isolates was significantly higher (P < 0.0001) than that in clinical isolates. Also, a three-codon deletion in inlA associated with a hyperinvasive phenotype was more prevalent in genomes from clinical isolates (primarily of clonal complex 6, serogroup 4b) than in those from food isolates (P < 0.001). In contrast, SSI-1 was significantly overrepresented (P < 0.001) in genomes from food isolates. We propose the hypothesis that SSI-1 and inlA play a role in the evolution of Canadian L. monocytogenes strains into either a virulent (represented by serogroup 4b clinical isolates) or an environmentally persistent (represented by serogroup 1/2a food isolates) phenotype. The combined presence of SSI-1 and inlA PMSC mutations have potential for use as genetic markers for risk assessment when L. monocytogenes is recovered from foods, indicating low potential for pathogenesis.

Determination of antimicrobial effect of protamine by transmission electron microscopy and SDS PAGE on Pseudomonas aeruginosa isolates from diabetic foot infection.

OBJECTIVES: Diabetic foot infection is one of the major complications of diabetes leading to lower limb amputations. Isolation and identification of bacteria causing diabetic foot infection, determination of antibiotic resistance, antimicrobial potential of protamine by electron microscopy and SDS-PAGE analysis, arethe aims of this study. MATERIALS AND METHODS: 285 pus samples from diabetic foot infection patients were collected from different hospitals of Karachi and Capital Health Hospital, Halifax, Canada. Clinical history of each patient was recorded. Bacterial isolates were cultured on appropriate media; identification was done by morphology, cultural and biochemical tests. Effect of protamine against multi drug resistant strains of Pseudomona aeruginosa was checked by minimum inhibitory concentration in 96 well micro-titer plates. The isolates were grown in bactericidal concentration of protamine on plates to isolate mutants. Effect of protamine on protein expression was checked by SDS- PAGE and ultra-structural morphological changes by transmission electron microscopy. RESULTS: Results indicated prevalence of foot infection as 92% in diabetic patients. Major bacterial isolates were Staphylococcus aureus 65 (23%), P. aeruginosa 80 (28.1%), Klebsiella spp. 37 (13%), Proteus mirabilis 79 (27.7%), and Escherichia coli 24 (12%). These isolates were highly resistant to different antibiotics. MIC value of protamine was 500 µg/ml against P. aeruginosa. SDS-PAGE analysis revealed that protamine can suppress expression of various virulence proteins and electron micrographs indicated condensation of cytoplasm and accumulation of protamine in cytoplasm without damaging the cell membrane. CONCLUSION: P. aeruginosa and S. aureus were the major isolates expressing multi-drug resistance and protamine sulfate represented good antimicrobial potential.

In vitro genomic and proteomic evidence of a type IV pili-like structure in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is an intracellular γ-proteobacteria and the etiological agent of piscirickettsiosis, which causes massive economic losses in the Chilean salmon industry. The type IV pili (T4P) play an important role in adherence to host cell surfaces and bacterial pathogenicity. T4P contains a variable number of components, as predicted in P. salmonis genomes. However, no studies have determined if P. salmonis possesses T4P. The aims of this investigation were to identify T4P components in the P. salmonis type strain LF-89T, evaluate respective transcript expressions, and analyze the main putative T4P proteins using bioinformatics and proteomic approaches. Two main clusters of P. salmonis T4P genes were found. Expression of the pilA gene was upregulated at 4 h post-infection (hpi), while pilQ was upregulated 4 days post-infection. At 16 hpi, pilB and pilD were strongly upregulated. The PilA amino acid sequence analysis showed a conserved N-terminal domain and sequence motifs critical for T4P biosynthesis. MudPIT analysis revealed PilA in the P. salmonis LF-89T proteome, and TEM showed pili-like filamentous structures on the P. salmonis surface. These results strongly suggest the presence of a T4P-like structure in P. salmonis.

Molecular and Biochemical Methods Useful for the Epigenetic Characterization of Chromatin-Associated Proteins in Bivalve Molluscs.

Bivalve molluscs constitute a ubiquitous taxonomic group playing key functions in virtually all ecosystems, and encompassing critical commercial relevance. Along with a sessile and filter-feeding lifestyle in most cases, these characteristics make bivalves model sentinel organisms routinely used for environmental monitoring studies in aquatic habitats. The study of epigenetic mechanisms linking environmental exposure and specific physiological responses (i.e., environmental epigenetics) stands out as a very innovative monitoring strategy, given the role of epigenetic modifications in acclimatization and adaptation. Furthermore, the heritable nature of many of those modifications constitutes a very promising avenue to explore the applicability of epigenetic conditioning and selection in management and restoration strategies. Chromatin provides a framework for the study of environmental epigenetic responses. Unfortunately, chromatin and epigenetic information are very limited in most non-traditional model organisms and even completely lacking in most environmentally and ecologically relevant organisms. The present work aims to provide a comprehensive and reproducible experimental workflow for the study of bivalve chromatin. First, a series of guidelines for the molecular isolation of genes encoding chromatin-associated proteins is provided, including information on primers suitable for conventional PCR, Rapid Amplification of cDNA Ends (RACE), genome walking and quantitative PCR (qPCR) experiments. This section is followed by the description of methods specifically developed for the analysis of histone and SNBP proteins in different bivalve tissues, including protein extraction, purification, separation and immunodetection. Lastly, information about available antibodies, their specificity and performance is also provided. The tools and protocols described here complement current epigenetic analyses (usually limited to DNA methylation) by incorporating the study of structural elements modulating chromatin dynamics.

A Syst-OMICS Approach to Ensuring Food Safety and Reducing the Economic Burden of Salmonellosis.

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance.

Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada.

Characterization and pathogenic role of outer membrane vesicles produced by the fish pathogen Piscirickettsia salmonis under in vitro conditions.

Piscirickettsia salmonis is one of the major fish pathogens affecting Chilean aquaculture. This Gram-negative bacterium is highly infectious and is the etiological agent of Piscirickettsiosis. Little is currently known about how the virulence factors expressed by P. salmonis are delivered to host cells. However, it is known that several Gram-negative microorganisms constitutively release outer membrane vesicles (OMVs), which have been implicated in the delivery of virulence factors to host cells. In this study, OMVs production by P. salmonis was observed during infection in CHSE-214 cells and during normal growth in liquid media. The OMVs were spherical vesicles ranging in size between 25 and 145 nm. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, the bacterial chaperonin Hsp60 was found in the OMVs of P. salmonis by Western-blot and LC-MS/MS analyses. Finally, in vitro infection assays showed that purified OMVs generated a cytopathic effect on CHSE-214 cells, suggesting a role in pathogenesis. Therefore, OMVs might be an important vehicle for delivering effector molecules to host cells during P. salmonis infection.

Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes.

Legionella pneumophila, a causative agent of Legionnaires' disease, is a facultative intracellular parasite of freshwater protozoa. Legionella pneumophila features a unique developmental network that involves several developmental forms including the infectious cyst forms. Reservoirs of L. pneumophila include natural and man-made freshwater systems; however, recent studies have shown that isolates of L. pneumophila can also be obtained directly from garden potting soil suggesting the presence of an additional reservoir. A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila. While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen. Using live DIC imaging coupled with focused transmission electron microscopy analyses, we report here that L. pneumophila is able to invade and establish Legionella-containing vacuoles (LCVs) in the intestinal cells. In addition, LCVs containing replicative and differentiated cyst forms were observed in the pseudocoelomic cavity and gonadal tissue of nematodes colonized with L. pneumophila. Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes. Our findings are novel as this is the first report, to our knowledge, of extraintestinal LCVs containing L. pneumophila cyst forms in C. elegans tissues, highlighting the potential of soil-dwelling nematodes as an alternate environmental reservoir for L. pneumophila.

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.

Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted.

Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells.

Legionella pneumophila is an intracellular bacterial parasite of freshwater protozoa and an accidental waterborne human pathogen. L. pneumophila is highly pleomorphic showing several forms that differentiate within its developmental cycle. In water, L. pneumophila produces viable but non-culturable cells (VBNCCs), which remain largely uncharacterized. We produced VBNCCs from two developmental forms of L. pneumophila [stationary phase forms (SPFs) and mature infectious forms (MIFs)] in two water microcosms [double-deionized (dd) and tap water] at 45°C. In contrast with SPFs, MIFs upheld a robust ultrastructure and high viability in the two water microcosms. In dd-water, MIFs and SPFs lost their culturability faster than in tap water and did not consume their poly-ß-hydroxybutyrate inclusions. Resuscitation in Acanthamoeba castellani was only possible for VBNCCs produced from SPFs in tap water. Addition of salts to dd-water prolonged L. pneumophila culturability to tap water levels, suggesting that L. pneumophila requires ions to maintain its readiness to resume growth. VBNCCs resisted detergent lysis and digestion in the ciliate Tetrahymena, except for VBNCCs produced from SPFs in dd-water. L. pneumophila VBNCCs thus show distinct traits according to its originating developmental form and the surrounding water microcosm.

The many forms of a pleomorphic bacterial pathogen-the developmental network of Legionella pneumophila.

Legionella pneumophila is a natural intracellular bacterial parasite of free-living freshwater protozoa and an accidental human pathogen that causes Legionnaires' disease. L. pneumophila differentiates, and does it in style. Recent experimental data on L. pneumophila's differentiation point at the existence of a complex network that involves many developmental forms. We intend readers to: (i) understand the biological relevance of L. pneumophila's forms found in freshwater and their potential to transmit Legionnaires' disease, and (ii) learn that the common depiction of L. pneumophila's differentiation as a biphasic developmental cycle that alternates between a replicative and a transmissive form is but an oversimplification of the actual process. Our specific objectives are to provide updates on the molecular factors that regulate L. pneumophila's differentiation (Section The Differentiation Process and Its Regulation), and describe the developmental network of L. pneumophila (Section Dissecting Lp's Developmental Network), which for clarity's sake we have dissected into five separate developmental cycles. Finally, since each developmental form seems to contribute differently to the human pathogenic process and the transmission of Legionnaires' disease, readers are presented with a challenge to develop novel methods to detect the various L. pneumophila forms present in water (Section Practical Implications), as a means to improve our assessment of risk and more effectively prevent legionellosis outbreaks.

The progeny of Legionella pneumophila in human macrophages shows unique developmental traits.

The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of amoebae and an accidental human pathogen that causes a noncommunicable atypical pneumonia known as Legionnaires' disease (LD). In some mammalian cells (e.g. HeLa), L. pneumophila follows a biphasic developmental cycle, differentiating between a replicative form that actively multiplies intracellularly, and a mature infectious form (MIF) that emerges as progeny. To date, it is not known whether the L. pneumophila progenies that emerge from amoebae and human macrophages reach similar developmental stages. Here, we demonstrate that in relation to the fully differentiated and highly infectious MIFs that emerge from amoebae, the L. pneumophila progeny that emerges from macrophages is morphologically undifferentiated, less resistant to antibiotics and less able to initiate infections. However, the L. pneumophila progeny from macrophages did not show any defects in intracellular growth. We thus concluded that macrophage infection with L. pneumophila yields a low number of bona fide MIFs. Because MIFs are the transmissive forms of L. pneumophila produced in vivo, our results showing that they are not efficiently produced in cultured macrophages provide an initial insight into why LD is not communicable.

Electron microscopy of legionella and legionella-infected cells.

Those investigators who study the morphology of Legionella and Legionella-infected cells have greatly benefited from the superior resolution afforded by electron microscopy (EM). It can also be said with confidence that EM will continue to reveal as yet to be discovered features of this fascinating intracellular pathogen. In this chapter we detail our practical experience in the application of three transmission electron microscopy (TEM) techniques to the study of Legionella: conventional ultrastructural analysis, immuno-gold labeling, and negative staining. Each of these techniques has particular, well-defined applications, which are discussed in the context of our in-house developed methods. We invite researchers to try the methods given here in the study of Legionella, and adopt TEM as part of their research tools arsenal.

The tetrahymena and acanthamoeba model systems.

Although the study of protozoology has been active for centuries, very few current academic curricula incorporate requirements or even options for coursework on the study of protists; yet, protozoa are becoming widely recognized by investigators as organisms that play a significant role in the evolution, pathogenicity, protection and amplification of human pathogens in the environment. This is particularly true for the study of Legionella, as this accidental human pathogen has naturally evolved to infect protozoa in fresh water environments. Researchers have made great progress in the study of pathogenicity, evolution, and ecology of Legionella and its protozoan hosts, which include amoebae and ciliated protozoa. Our own collaboration in this field has been active for over a decade, and we have gained a valuable experience working with these protozoa, particularly aspects of their biology and the methods needed to address new experimental concepts. Therefore, in this chapter we provide the most effective procedures that we have developed or modified through our years of practice. We also offer notes on what procedures, in our opinion, should be avoided; and we provide the rationale for such precautions.

Mechanism of invasion of lung epithelial cells by filamentous Legionella pneumophila.

Legionella, the aetiological agent responsible for Legionellosis, is an opportunistic pathogen that infects humans upon the inhalation of contaminated aerosolized water droplets. Legionella is pleomorphic and its different morphotypes exhibit varying degrees of virulence. While the filamentous forms of Legionella pneumophila (Lp) have been reported in patient samples since the first description of legionellosis, their role in disease has not been studied. Our results show that both E-cadherin and ß1 integrin receptors mediate filamentous Lp (FLp) attachment to lung epithelial cells (LECs). The activation of these receptors induces the formation of actin enriched membrane surface structures that we designated 'hooks' and 'membrane wraps'. These structures entrap the filaments on the cell surface leading to their gradual internalization through a zipper mechanism of phagocytosis dependent on actomyosin activity. The supply of E-cadherin receptors from the recycling pathway and ß1 integrins released from focal adhesion turnover are required to sustain this process. Intracellular FLp inhabits a vacuolar compartment where filaments differentiate into short rods and replicate to produce infective progeny. Here we are reporting a first description of the invasion mechanism used by FLp to invade LECs. Therefore, filamentous morphotype of Lp can induce its own uptake by LECs and has the potential ability to cause disease.