Audrey Chong

The Early Phagosomal Stage of Francisella tularensis Determines Optimal Phagosomal Escape and Francisella Pathogenicity Island Protein Expression

ABSTRACT Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.

Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.

The Francisella Intracellular Life Cycle: Toward Molecular Mechanisms of Intracellular Survival and Proliferation

The tularemia-causing bacterium Francisella tularensis is a facultative intracellular organism with a complex intracellular lifecycle that ensures its survival and proliferation in a variety of mammalian cell types, including professional phagocytes. Because this cycle is essential to Francisella pathogenesis and virulence, much research has focused on deciphering the mechanisms of its intracellular survival and replication and characterizing both bacterial and host determinants of the bacteriums intracellular cycle. Studies of various strains and host cell models have led to the consensual paradigm of Francisella as a cytosolic pathogen, but also to some controversy about its intracellular cycle. In this review, we will detail major findings that have advanced our knowledge of Francisella intracellular survival strategies and also attempt to reconcile discrepancies that exist in our molecular understanding of the Francisella–phagocyte interactions.

Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway.

Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival and proliferation.

The Francisella O-antigen mediates survival in the macrophage cytosol via autophagy avoidance.

Autophagy is a key innate immune response to intracellular parasites that promotes their delivery to degradative lysosomes following detection in the cytosol or within damaged vacuoles. Like Listeria and Shigella, which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen Francisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how Francisella evades autophagy, we screened a library of F. tularensis subsp. tularensis Schu S4 HimarFT transposon mutants in GFP‐LC3‐expressing murine macrophages by microscopy for clones localized within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at 6 h post‐infection, whose HimarFT insertions clustered to fourgenetic loci involved in lipopolysaccharidic and capsular O‐antigen biosynthesis. Consistent with the HimarFT mutants, in‐frame deletion mutants of two representative loci, FTT1236 and FTT1448c (manC), lacking both LPS and capsular O‐antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild‐type Francisella, the O‐antigen deletion mutants were ubiquitinated, and recruited the autophagy adaptor p62/SQSTM1 and LC3 prior to cytosolic clearance. Autophagy‐deficient macrophages partially supported replication of both mutants, indicating that O‐antigen‐lacking Francisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.

The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization

ABSTRACT A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular trafficking events of L. pneumophila, including the recruitment of mitochondria, cytoskeletal alterations, the inhibition of phagosome-lysosome fusion, and association with the endoplasmic reticulum. Microscopy and flow cytometry studies indicated that HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages and induced transient changes in the organization of actin microfilaments in CHO cells. Ectopic expression of HtpB in the cytoplasm of transfected CHO cells also led to modifications in actin microfilaments similar to those produced by HtpB-coated beads but did not change the distribution of mitochondria. Association of phagosomes containing HtpB-coated beads with the endoplasmic reticulum was not consistently detected by either fluorescence or electron microscopy studies, and only a modest delay in the fusion of TrOv-labeled lysosomes with phagosomes containing HtpB-coated beads was observed. HtpB is the first Legionella protein and the first chaperonin shown to, by means of our functional models, induce mitochondrial recruitment and microfilament rearrangements, two postinternalization events that typify the early trafficking of virulent L. pneumophila.

Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth

The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila.

Low Dose Vaccination with Attenuated Francisella tularensis Strain SchuS4 Mutants Protects against Tularemia Independent of the Route of Vaccination

Tularemia, caused by the Gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

ABSTRACT Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.

A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells

Type III secretion system 1 (T3SS1) is used by the enteropathogen Salmonella enterica serovar Typhimurium to establish infection in the gut. Effector proteins translocated by this system across the plasma membrane facilitate invasion of intestinal epithelial cells. One such effector, the inositol phosphatase SopB, contributes to invasion and mediates activation of the pro-survival kinase Akt. Following internalization, some bacteria escape from the Salmonella-containing vacuole into the cytosol and there is evidence suggesting that T3SS1 is expressed in this subpopulation. Here, we investigated the post-invasion role of T3SS1, using SopB as a model effector. In cultured epithelial cells, SopB-dependent Akt phosphorylation was observed at two distinct stages of infection: during and immediately after invasion, and later during peak cytosolic replication. Single cell analysis revealed that cytosolic Salmonella deliver SopB via T3SS1. Although intracellular replication was unaffected in a SopB deletion mutant, cells infected with ΔsopB demonstrated a lack of Akt phosphorylation, earlier time to death, and increased lysis. When SopB expression was induced specifically in cytosolic Salmonella, these effects were restored to levels observed in WT infected cells, indicating that the second wave of SopB protects this infected population against cell death via Akt activation. Thus, T3SS1 has two, temporally distinct roles during epithelial cell colonization. Additionally, we found that delivery of SopB by cytosolic bacteria was translocon-independent, in contrast to canonical effector translocation across eukaryotic membranes, which requires formation of a translocon pore. This mechanism was also observed for another T3SS1 effector, SipA. These findings reveal the functional and mechanistic adaptability of a T3SS that can be harnessed in different microenvironments.