Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions

Elucidation of molecular mechanisms underlying hostpathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.     

Incoming pathogens team up with harmless 'resident' bacteria

Microbial diseases occur as a result of multifarious host-pathogen interactions. However, invading pathogens encounter a large number of different harmless and beneficial bacterial species, which colonize and reside in the host. Surprisingly, there has been little study of the possible interactions between incoming pathogens and the resident bacterial community. Recent studies have revealed that resident bacteria assist different types of incoming pathogens via a wide variety of mechanisms including cell-cell signaling, metabolic interactions, evasion of the immune response and a resident-to-pathogen switch. This calls for serious consideration of pathogen-microbe interactions in the host with respect to disease severity and progression.     

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

RNA profiling in host-pathogen interactions

The development of novel anti-bacterial treatment strategies will be aided by an increased understanding of the interactions that take place between bacteria and host cells during infection. Global expression profiling using microarray technologies can help to describe and define the mechanisms required by bacterial pathogens to cause disease and the host responses required to defeat bacterial infection.     

The molecular basis of fibronectin-mediated bacterial adherence to host cells

Many pathogenic Gram-positive bacteria produce cell wall-anchored proteins that bind to components of the extracellular matrix (ECM) of the host. These bacterial MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are thought to play a critical role in infection. One group of MSCRAMMs, produced by staphylococci and streptococci, targets fibronectin (Fn, a glycoprotein found in the ECM and body fluids of vertebrates) using repeats in the C-terminal region of the bacterial protein. These bacterial Fn-binding proteins (FnBPs) mediate adhesion to host tissue and bacterial uptake into non-phagocytic host cells. Recent studies on interactions between the host and bacterial proteins at the residue-specific level and on the mechanism of host cell invasion are providing a much clearer picture of these processes.     

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1α, −6, −7, −8, −18, TNF-α and GM-CSF, but not TGF-β or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen–host cell interactions.     

No effect of Wolbachia on resistance to intracellular infection by pathogenic bacteria in Drosophila melanogaster

Multiple studies have shown that infection with the endosymbiotic bacterium Wolbachia pipientis confers Drosophila melanogaster and other insects with resistance to infection by RNA viruses. Studies investigating whether Wolbachia infection induces the immune system or confers protection against secondary bacterial infection have not shown any effect. These studies, however, have emphasized resistance against extracellular pathogens. Since Wolbachia lives inside the host cell, we hypothesized that Wolbachia might confer resistance to pathogens that establish infection by invading host cells. We therefore tested whether Wolbachia-infected D. melanogaster are protected against infection by the intracellular pathogenic bacteria Listeria monocytogenes and Salmonella typhimurium, as well as the extracellular pathogenic bacterium Providencia rettgeri. We evaluated the ability of flies infected with Wolbachia to suppress secondary infection by pathogenic bacteria relative to genetically matched controls that had been cured of Wolbachia by treatment with tetracycline. We found no evidence that Wolbachia alters host ability to suppress proliferation of any of the three pathogenic bacteria. Our results indicate that Wolbachia-induced antiviral protection does not result from a generalized response to intracellular pathogens.     

A model of bacterial intestinal infections in Drosophila melanogaster

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.     

Mechanisms of outer membrane vesicle entry into host cells

Bacterial outer membrane vesicles (OMVs) are nano‐sized compartments consisting of a lipid bilayer that encapsulates periplasm‐derived, luminal content. OMVs, which pinch off of Gram‐negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV‐host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies.     

From cellular microbiology to bacteria‐based next generations of cancer immunotherapies

Pioneer work by Prof. Cossart among others, studying the interactions between pathogenic bacteria and host cells (this discipline was termed Cellular Microbiology), was fundamental to determine the bacterial infection processes and to improve our knowledge of different cellular mechanisms. The study of bacteria–host interactions also involves in vivo host immune responses, which can be manipulated by bacteria, being these last potent tools for different immunotherapies. During the last years, tumour immunotherapies, mainly the use of antibodies that target immune checkpoints [checkpoint inhibitors (CPI)], have been a revolution in oncology, allowing the treatment of tumours otherwise with very bad prognosis. In the same direction, bacteria inoculations have been used from long to treat some cancers; for example, non‐muscle‐invasive bladder cancer can be successfully treated with the bacterium Bacillus Calmette Guerin (BCG). More recently, it has been shown that microbiota could determine the success of CPI immunotherapies and intense research is being performed in order to use bacteria as immunotherapy tools due to their ability to activate the immune system. In this context, to expand the knowledge of the bacteria–immune system interactions will be fundamental to improve tumour immunotherapies.     

Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

Targeted Proteomics for Studying Pathogenic Bacteria

Mass spectrometry‐based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria–host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS‐based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.     

Intracellular bacterial communities of uropathogenic Escherichia coli in urinary tract pathogenesis

Urinary tract infections in young, healthy women frequently recur, despite their traditional classification as acute infections. Conventional wisdom dictates that uropathogens causing recurrent infections in such individuals come from the fecal or vaginal flora, in the same manner as the initial infection. However, recent studies of uropathogenic Escherichia coli have found that it can carry out a complex developmental program within the superficial epithelial cells of the mouse bladder, forming intracellular bacterial communities with many biofilm-like properties. These intracellular biofilms allow the bacteria to outlast a strong host immune response to establish a dormant reservoir of pathogens inside the bladder cells. Re-emergence of bacteria from this reservoir might be the source of recurrent infection.     

A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to non-host specialized pathogens

With the emergence of multiply resistant Staphylococcus aureus, there is an urgent need to better understand the molecular determinants of S. aureus pathogenesis. A model of staphylococcal pathogenesis in zebrafish embryos has been established, in which host phagocytes are able to mount an effective immune response, preventing overwhelming infection from small inocula. Myeloid cell depletion, by pu.1 morpholino-modified antisense injection, removes this immune protection. Macrophages and neutrophils are both implicated in this immune response, phagocytosing circulating bacteria. In addition, in vivo phagocyte/bacteria interactions can be visualized within transparent embryos. A preliminary screen for bacterial pathogenesis determinants has shown that strains bearing mutations in perR, pheP and saeR are attenuated. perR and pheP mutants are deficient in growth in vivo, and their virulence is not fully restored by myeloid cell depletion. On the other hand, saeR mutants are able to grow in vivo, and are completely restored to virulence by myeloid cell depletion. Thus specific pathogen gene function can be matched with particular facets of host response. Zebrafish are a new addition to the tools available for the study of S. aureus pathogenesis, and may provide insights into the interactions of bacterial and host genomes in determining the outcome of infection.     

Living in the danger zone: innate immunity to Salmonella

Phagocytic cells, including macrophages, neutrophils and dendritic cells, are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium. These cells can have several roles during the early stage of an infection including controlling bacterial replication and producing cytokines and chemokines that activate and recruit additional cells. Macrophages, neutrophils and dendritic cells increase in number early after oral Salmonella infection and produce cytokines important in host survival such as tumor necrosis factor alpha (TNF-alpha). All three phagocytic cell types also harbor bacteria during infection. Natural killer cells, natural killer T cells and T cell receptor alpha beta T cells also respond rapidly to infection and are early sources of interferon-gamma during infection with Salmonella. Studies using infection models with Salmonella are providing a picture of the innate response to bacteria and insight into the role of defined cell types and cytokines important in the transition from innate to adaptive immunity.