Phagosomal Environment Research Articles

Dissociation of innate immune responses in microglia infected with Listeria monocytogenes

Microglia, the innate immune cells of the brain, plays a central role in cerebral listeriosis. Here, we present evidence that microglia control Listeria infection differently than macrophages. Infection of primary microglial cultures and murine cell lines with Listeria resulted in a dual function of the two gene expression programmes involved in early and late immune responses in macrophages. Whereas the bacterial gene hly seems responsible for both transcriptional programmes in macrophages, Listeria induces in microglia only the tumor necrosis factor (TNF)-regulated transcriptional programme. Listeria also represses in microglia the late immune response gathered in two clusters, microbial degradation, and interferon (IFN)-inducible genes. The bacterial gene actA was required in microglia to induce TNF-regulated responses and to repress the late response. Isolation of microglial phagosomes revealed a phagosomal environment unable to destroy Listeria. Microglial phagosomes were also defective in several signaling and trafficking components reported as relevant for Listeria innate immune responses. This transcriptional strategy in microglia induced high levels of TNF-α and monocyte chemotactic protein-1 and low production of other neurotoxic compounds such as nitric oxide, hydrogen peroxide, and Type I IFNs. These cytokines and toxic microglial products are also released by primary microglia, and this cytokine and chemokine cocktail display a low potential to trigger neuronal apoptosis. This overall bacterial strategy strongly suggests that microglia limit Listeria inflammation pattern exclusively through TNF-mediated responses to preserve brain integrity. GLIA 2014;62:233–246

Mycobacterium tuberculosis Responds to Chloride and pH as Synergistic Cues to the Immune Status of its Host Cell

The ability of Mycobacterium tuberculosis (Mtb) to thrive in its phagosomal niche is critical for its establishment of a chronic infection. This requires that Mtb senses and responds to intraphagosomal signals such as pH. We hypothesized that Mtb would respond to additional intraphagosomal factors that correlate with maturation. Here, we demonstrate that [Cl−] and pH correlate inversely with phagosome maturation, and identify Cl− as a novel environmental cue for Mtb. Mtb responds to Cl− and pH synergistically, in part through the activity of the two-component regulator phoPR. Following identification of promoters responsive to Cl− and pH, we generated a reporter Mtb strain that detected immune-mediated changes in the phagosomal environment during infection in a mouse model. Our study establishes Cl− and pH as linked environmental cues for Mtb, and illustrates the utility of reporter bacterial strains for the study of Mtb-host interactions in vivo.

Proteomics reveals dynamic phagosomal environment during Mycobacterium -HIV co-infection

Event Abstract Back to Event Proteomics reveals dynamic phagosomal environment during Mycobacterium -HIV co-infection Rakesh Ganji1, Snigdha Dhali2, Arshad Rizvi1, Srikanth Rapole2 and Sharmistha Banerjee1* 1 University of Hyderabad, India 2 National Centre for Cell Science, India Mycobacterial infection is a major cause for increased mortality in HIV infected patients. In order to understand the coalition between Mycobacteria and HIV that makes these diseases more destructive when confronted together, we undertook a proteomics approach to identify differentially regulated proteins during intra-phagosomal phase of infection in macrophages under mycobacteria-HIV co-infection. A comparative protein expression pattern of host proteins during mycobacterial and mycobacteria-HIV co-infection of macrophages revealed 95 phagosomal and 70 mitochondrial proteins differentially regulated. An extreme cytoskeletal rearrangement was observed with categorical up-regulation of energy metabolism proteins during co-infection. Purinergic receptors, which are capable of adjusting cellular metabolic needs and also help HIV entry, were observed to be upregulated during co-infection. Interestingly, co-infected host cells mounted augmented inflammatory response, as evident from increased manganese superoxide dismutase and intracellular ROS levels. 94 mycobacterial proteins were differentially regulated upon co-infection, majority of which belonged to intermediary metabolism, respiration, cell wall synthesis and information pathways. During co-infection, Mycobacteria showed increased expression of proteins of ESX secretion systems that are instrumental in secretion of virulent proteins. An increased expression of cAMP dependent regulator (Rv2565), Rv1265 (regulated by cAMP dependent regulator) and RecF (DNA repair protein) upon co-infection may protect mycobacteria from increased host ROS levels. Altered expression of toxin-antitoxin family of proteins during mono- and co-infection suggested regulated mycobacterial stress response that may influence programmed cell death. Conclusively, proteomics data suggested a dynamic host-pathogen environment during co-infection leading to identification of critical factors that explained the coalition between HIV and mycobacteria. Keywords: Mycobacterium, HIV, Co-infection, phagosome, Proteomics Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Ganji R, Dhali S, Rizvi A, Rapole S and Banerjee S (2013). Proteomics reveals dynamic phagosomal environment during Mycobacterium -HIV co-infection. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00822 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sharmistha Banerjee, University of Hyderabad, Hyderabad, India, sbsl@uohyd.ernet.in Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rakesh Ganji Snigdha Dhali Arshad Rizvi Srikanth Rapole Sharmistha Banerjee Google Rakesh Ganji Snigdha Dhali Arshad Rizvi Srikanth Rapole Sharmistha Banerjee Google Scholar Rakesh Ganji Snigdha Dhali Arshad Rizvi Srikanth Rapole Sharmistha Banerjee PubMed Rakesh Ganji Snigdha Dhali Arshad Rizvi Srikanth Rapole Sharmistha Banerjee Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Measurement of phagocytosis, phagosome acidification, and intracellular killing of Staphylococcus aureus.

Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe several methods to assess important properties of phagosomes in macrophages, such as phagocytosis, acidification of the phagosome contents during the maturation process, and the ability of phagosomes to inactivate and kill pathogens. Phagocytosis and phagosome acidification assays are FACS-based assays where labeled bacteria are used as probes to monitor internalization into a phagosome and to detect the pH of the phagosome environment. The killing assay is based on the counting of bacterial colonies after recovery of internalized bacteria from macrophages.

A Method for Spatially Resolved Local Intracellular Mechanochemical Sensing and Organelle Manipulation

Because both the chemical and mechanical properties of living cells play crucial functional roles, there is a strong need for biophysical methods to address these properties simultaneously. Here we present a novel (to our knowledge) approach to measure local intracellular micromechanical and chemical properties using a hybrid magnetic chemical biosensor. We coupled a fluorescent dye, which serves as a chemical sensor, to a magnetic particle that is used for measurement of the viscoelastic environment by studying the response of the particle to magnetic force pulses. As a demonstration of the potential of this approach, we applied the method to study the process of phagocytosis, wherein cytoskeletal reorganization occurs in parallel with acidification of the phagosome. During this process, we measured the shear modulus and viscosity of the phagosomal environment concurrently with the phagosomal pH. We found that it is possible to manipulate phagocytosis by stalling the centripetal movement of the phagosome using magnetic force. Our results suggest that preventing centripetal phagosomal transport delays the onset of acidification. To our knowledge, this is the first report of manipulation of intracellular phagosomal transport without interfering with the underlying motor proteins or cytoskeletal network through biochemical methods.

The Mycobacterium tuberculosis SecA2 System Subverts Phagosome Maturation To Promote Growth in Macrophages

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.

Global changes in gene expression by the opportunistic pathogen Burkholderia cenocepacia in response to internalization by murine macrophages

BackgroundBurkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation.ResultsWe examined gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) combined with microarray analysis. We identified 767 genes with significantly different levels of expression by intracellular bacteria, of which 330 showed increased expression and 437 showed decreased expression. Affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. In general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affected bacterial entry into macrophages and intracellular replication. We also show that intracellular B. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on SCOTS-identified genes. In particular, genes involved in bacterial motility, cobalamin biosynthesis, the type VI secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of B. cenocepacia.ConclusionsB. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. The analysis of the transcriptomic response of intracellular B. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of B. cenocepacia in macrophages. This adaptive response does not require the expression of any specific virulence-associated factor, which is consistent with the opportunistic nature of this microorganism. Further investigation into the remaining SCOTS-identified genes will provide a more complete picture of the adaptive response of B. cenocepacia to the host cell environment.

Escape from the Phagosome: The Explanation for MHC-I Processing of Mycobacterial Antigens?

Mycobacterium tuberculosis (Mtb) is thought to live in an altered phagosomal environment. In this setting, the mechanisms by which mycobacterial antigens access the major histocompatibility class I (MHC-I) processing machinery remain incompletely understood. There is evidence that Mtb antigens can be processed in both endocytic and cytosolic environments, with different mechanisms being proposed for how Mtb antigens can access the cytosol. Recently, electron microscopy was used to demonstrate that Mtb has the potential to escape the phagosome and reside in the cytosol. This was postulated as the primary mechanism by which Mtb antigens enter the MHC-I processing and presentation pathway. In this commentary, we will review data on the escape of Mtb from the cytosol and whether this escape is required for antigen presentation to CD8+ T cells.

The Acid Phosphatase AcpA Is SecretedIn Vitroand in Macrophages by Francisella spp

Francisella tularensis is a remarkably infectious facultative intracellular pathogen that causes the zoonotic disease tularemia. Essential to the pathogenesis of F. tularensis is its ability to escape the destructive phagosomal environment and inhibit the host cell respiratory burst. F. tularensis subspecies encode a series of acid phosphatases, which have been reported to play important roles in Francisella phagosomal escape, inhibition of the respiratory burst, and intracellular survival. However, rigorous demonstration of acid phosphatase secretion by intracellular Francisella has not been shown. Here, we demonstrate that AcpA, which contributes most of the F. tularensis acid phosphatase activity, is secreted into the culture supernatant in vitro by F. novicida and F. tularensis subsp. holarctica LVS. In addition, both F. novicida and the highly virulent F. tularensis subsp. tularensis Schu S4 strain are able to secrete and also translocate AcpA into the host macrophage cytosol. This is the first evidence of acid phosphatase translocation during macrophage infection, and this knowledge will greatly enhance our understanding of the functions of these enzymes in Francisella pathogenesis.

Multiparameter flow cytometric kinetics of phagocyte stimulus responses

THE recently published Technical Note by Vines et al., ‘‘A Flow-Cytometric Method for Continuous Measurement of Intracellular Ca Concentration,’’ Cytometry Part A 77A:1091 1097, 2010, demonstrated that such measurements are possible in an Accuri C6 flow cytometer. Their contribution demonstrates that rapid measurements can be achieved when stimuli are added to cells (in an open tube) which then flow via a pump mechanism, yielding observations within 5 s, judging from their figures, albeit without thermostating or stirring the sample. Intracellular kinetic measurements of cytoplasmic Ca (and other events) using other flow cytometers (FACS 440, MoFlo, Aria) have been previously reported by a number of laboratories, including ours and extensively reviewed (1 9). Since our own first report in 1986 (10), we have published numerous flow cytometric measurements of cytoplasmic Ca fluxes ([Ca]in) in various blood cells, using a modified FACS 440 (BD) or MoFlo (Cytomation), injecting the compounds into a stirred thermostated flowing cell suspension. These flow cytometers, and the LSRII and FACSAria to which we are now adapting the sample handling system we described, use N2 pressure to move the cell stream, which permits the sample to be stoppered and reduces risk from potential biohazards. Thermostating and stirring assure physiologic conditions and rapid mixing. Injecting into pressure-driven, flowing cells a very short (<10 cm) distance from the nozzle, permits continuous observation of cellular responses within 4 s after reagent addition without using high flow rates. Using the technique above, we also have correlated cytoplasmic changes in Ca and pH, and consequent reactive oxygen and lytic entities, with specific receptor occupancy and subpopulation responses by and with the neutrophils’ receptor cross-linking requirements (11). Similar studies performed with monocytes (12) permitted differentiation between cells responding and not responding withD[Ca] by the presence or absence of a specific receptor (CD14) on their surface (Fig. 1). Our flow cytometric methods have now been applied to studies of the early kinetics of cytoplasmic and phagosomal changes as cells respond to stimulating organisms (e.g., changes in phagosomal pH, generation of reactive oxygen species (ROS), presence of lytic enzymes) by labeling the phagocytized entity so that it acts as an indicator of conditions in that compartment (11). Recently, we showed that neutrophil (PMN) stimulation by Staphylococcus epidermidis (13) requires opsonization of the organism. A transient spike in D[Ca]in is maximal within 18 s and originates from intra PMN stores. Using pHrodo (LifeSciences), we further showed that the phagosome enclosing S. epidermidis becomes increasingly alkaline, in contrast to the acidification we observed when a fungus, Cryptococcus neoformans, was phagocytized. By using flow cytometry and our adaptation for rapid kinetics (described above) in conjunction with multiple labels on the same organism, we were able to correlate the initial D[Ca]in signal not only with the phagosomal pH (pHp) but also with the ROS, generated in the phagosome and the receptor occupancy on the same PMN. Quenching the fluorescence of bound but not yet phagosome-enclosed S. epidermidis permitted us to focus on the conditions within it and to begin investigation of the role of extracellular Ca. This method gains greater potential relevance when it is used to investigate the very early, coordinated signaling events that occur upon binding and phagocytosis of organisms like mycobacteria that are able to alter the phagocyte response to evade killing and establish latent infection. Binding-induced fluxes resulting in rapid changes in local ion concentrations and their relationships to activation of membrane-localized

Regulation of the Edwardsiella ictaluri Type III Secretion System by pH and Phosphate Concentration through EsrA, EsrB, and EsrC

A recently described Edwardsiella ictaluri type III secretion system (T3SS) with functional similarity to the Salmonella pathogenicity island 2 T3SS is required for replication in channel catfish head-kidney-derived macrophages (HKDM) and virulence in channel catfish. Quantitative PCR and Western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Mutagenesis studies demonstrated that expression is under the control of the EsrAB two-component regulatory system. EsrB also induces upregulation of the AraC-type regulatory protein EsrC, which enhances expression of the EscB/EseG chaperone/effector operon in concert with EsrB and induces expression of the pEI1-encoded effector, EseH. EsrC also induces expression of a putative type VI secretion system translocon protein, EvpC, which is secreted under the same low-pH conditions as the T3SS translocon proteins. The pEI2-encoded effector, EseI, was upregulated under low-pH and low-phosphate conditions but not in an EsrB- or EsrC-dependent manner. Mutations of EsrA and EsrB both resulted in loss of the ability to replicate in HKDM and full attenuation in the channel catfish host. Mutation of EsrC did not affect intracellular replication but did result in attenuation in catfish. Although EsrB is the primary transcriptional regulator for E. ictaluri genes within the T3SS pathogenicity island, EsrC regulates expression of the plasmid-carried effector eseH and appears to mediate coordinated expression of the T6SS with the T3SS.

Virulence-related Mycobacterium avium subsp hominissuis MAV_2928 gene is associated with vacuole remodeling in macrophages

BackgroundMycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.ResultsMAV_2928 gene is primarily upregulated upon phagocytosis. The transcriptional profile of macrophages infected with the wild-type bacterium and the mutant were examined using DNA microarray, which showed that the two bacteria interact uniquely with mononuclear phagocytes. Based on the results, it was hypothesized that the phagosome environment and vacuole membrane of the wild-type bacterium might differ from the mutant. Wild-type bacterium phagosomes expressed a number of proteins different from those infected with the mutant. Proteins on the phagosomes were confirmed by fluorescence microscopy and Western blot. The environment in the phagosome of macrophages infected with the mutant differed from the environment of vacuoles with M. hominissuis wild-type in the concentration of zinc, manganese, calcium and potassium.ConclusionThe results suggest that the MAV_2928 gene/operon might participate in the establishment of bacterial intracellular environment in macrophages.

Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry

Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies.

Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis

Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pH(p)) and oxidative burst, on a cell-by-cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis (Se). The availability of a new, highly sensitive pH probe, pHrodo, permits observation of increasing pH(p). Simultaneous labeling of the organism, applicable to any phagocyte target, with a probe insensitive to pH and oxidative species, such as AlexaFluor350, permits distinction between binding and functional responses to it by ratioing fluorescences. Addition of an extracellular-specific quencher (Trypan blue) permits distinction between bound and phagosome-enclosed targets, so that conditions within the closed phagosome can be studied. We found that opsonization is required for functional activation of PMN by Se, that the organism causes early alkalinization of the phagosome (in contrast to Cf which hyperacidifies it), and that extracellular Ca(2+) is not required for cytoplasmic Ca(2+) signaling but contributes markedly to binding of Se to PMN and to ensuant bactericidal functions. These findings lead to a new approach to the study of select organisms, like Cf and Mtb, which evade killing by manipulating the phagosomal environment.

Characterisation of porin genes from Mycobacterium fortuitumand their impact on growth

BackgroundHighly pathogenic mycobacteria like Mycobacterium tuberculosis are characterised by their slow growth and their ability to reside and multiply in the very hostile phagosomal environment and a correlation between the growth rate of mycobacteria and their pathogenicity has been hypothesised. Here, porin genes from M. fortuitum were cloned and characterised to address their impact on the growth rate of fast-growing and pathogenic mycobacteria.ResultsTwo genes encoding porins orthologous to MspA from M. smegmatis, porM1 and porM2, were cloned from M. fortuitum strains, which were originally isolated from human patients. Both porin genes were at least partially able to complement the mutations of a M. smegmatis mutant strain lacking the genes mspA and mspC with respect to the growth rate. PorM1 and porM2 were present in different strains of M. fortuitum including the type strain. Comparative expression analysis of porM genes revealed divergent porin expression among analysed M. fortuitum strains. Repression of the expression of porins by antisense technique decreased the growth rates of different M. fortuitum. The effects of over-expression of porM1 as well as porM2 varied depending on the strain and the concentration of antibiotic added to the medium and indicated that PorM1 and PorM2 enhance the growth of M. fortuitum strains, but also the diffusion of the antibiotic kanamycin into the cells.ConclusionThis study demonstrates the important role of porin expression in growth as well as antibiotic susceptibility of the opportunistic bacterium M. fortuitum.

Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells

Intramacrophage survival appears to be a pathogenic trait common to Salmonellae and definition of the metabolic requirements of Salmonella within macrophages might provide opportunities for novel therapeutic interventions. We show that loss of PurG function in Salmonella enterica serovar Typhimurium SL1344 leads to death of the bacterium in RAW264.7 cells, which was due to unavailability of purine nucleotides but not thiamine in the phagosome of RAW264.7 cells. Phagosomal escape of purG mutant restored growth, suggesting that the phagosomal environment, but not the cytosol, is toxic to Salmonella purine auxotrophs. NADPH oxidase inhibition restored the growth of purG mutant in RAW264.7 cells, implying that the Salmonella-containing vacuole acquires reactive oxygen species (ROS) that are lethal to purine auxotrophs. Under purine limiting conditions, purG mutant was unable to repair the damage caused by hydrogen peroxide or UV irradiation, suggesting that ROS-mediated DNA damage may have been responsible for the attenuated phenotype of purG mutant in RAW264.7 cells and in mice. These studies highlight the possibility of utilizing the Salmonella purine nucleotide biosynthetic pathway as a prospective therapeutic target and also underline the importance of metabolic pathways in assembling a comprehensive understanding of the host-pathogen interactions inside phagocytic cells.

Salmonella sensing of anti‐microbial mechanisms to promote survival within macrophages

Salmonella enterica is a facultative intracellular pathogen that replicates within macrophages. The interaction of this pathogen with mammalian cells is a complex process involving hundreds of bacterial products that are sensed by and alter mammalian hosts. Numerous bacterial genes and their protein products have been identified that are required for Salmonella to resist killing by host innate immunity and to modify host processes. Many of these genes are regulated by a specific bacterial sensor, the PhoQ protein, which responds to the acidified phagosome environment. PhoQ is a sensor histidine kinase, which when activated in vivo within acidified macrophage phagosomes, regulates cell surface modifications that promote resistance to antimicrobial peptides and oxidative stress, alter the phagosome to promote intracellular survival, and reduce innate immune recognition. In this review, we discuss mechanisms by which Salmonella interacts with macrophages and focus in detail on recent reports describing the role of antimicrobial peptides and pH in PhoQ activation.

Did Bacterial Sensing of Host Environments Evolve from Sensing within Microbial Communities?

Bacteria sense and respond to their environment, enabling adaptation to diverse niches, including multicellular eukaryotes. In this issue of Cell Host & Microbe, Torres et al. describe how the bacterium Staphylococcus aureus responds to heme as a molecular marker of the mammalian host environment. It is likely that mechanisms for sensing such markers evolved from systems that recognized cues present in microbial communities before the emergence of eukaryotes.

Targeted Protein Degradation by Salmonella under Phagosome-mimicking Culture Conditions Investigated Using Comparative Peptidomics

The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Because of the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g. contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study S. enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal environment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified and quantified 5,163 peptides originating from 682 proteins, and the data clearly indicated that compared with Salmonella cultured in the rich medium, cells cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed using traditional, bottom-up proteomic methods, and when the peptidomics and proteomics data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

Activation of the Bacterial Sensor Kinase PhoQ by Acidic pH

The Salmonellae PhoQ sensor kinase senses the mammalian phagosome environment to activate a transcriptional program essential for virulence. The PhoQ periplasmic domain binds divalent cations, forming bridges with inner membrane phospholipids to maintain PhoQ repression. PhoQ also binds and is activated by cationic antimicrobial peptides. In this work, PhoQ is directly activated by exposure of the sensor domain to pH 5.5. NMR spectroscopy indicates that at acidic pH, the PhoQ periplasmic domain adopts a conformation different from that in the presence of divalent cations or antimicrobial peptides. The conformation is partially simulated by mutation of histidine 157, which is part of an interaction network that distinguishes the repressed conformation. The effects of antimicrobial peptides and pH on PhoQ activity are additive. We propose a model of activation by antimicrobial peptides via disruption of the cation bridges and/or by acidification of the periplasm through destabilization of the interaction network.