Publications

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag.

Francisella tularensis is a gram-negative facultative bacterium that causes the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. In order to help understand the mechanisms by which this occurs, we performed Affymetrix microarray analysis on transcripts from blood monocytes infected with the virulent Type A Schu S4 strain. Results showed that expression of several host response genes were reduced such as those associated with interferon signaling, Toll-like receptor signaling, autophagy and phagocytosis. When compared to microarrays from monocytes infected with the less virulent F. tularensis subsp. novicida, we found qualitative differences and also a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes in the Schu S4 strain. Notably, the PI3K/Akt1 pathway appeared specifically down-regulated following Schu S4 infection and a concomitantly lower cytokine response was observed. This study identifies several new factors potentially important in host cell subversion by the virulent Type A F. tularensis that may serve as novel targets for drug discovery.

Tularemia is a zoonotic disease caused by the Gram-negative intracellular pathogen Francisella tularensis. These bacteria evade phagolysosomal fusion, escape from the phagosome and replicate in the host cell cytoplasm. IFNgamma has been shown to suppress the intra-macrophage growth of Francisella through both nitric oxide-dependent and -independent pathways. Since Francisella is known to subvert host immune responses, we hypothesized that this pathogen could interfere with IFNgamma signaling. Here, we report that infection with Francisella suppresses IFNgamma-induced STAT1 expression and phosphorylation in both human and murine mononuclear phagocytes. This suppressive effect of Francisella is independent of phagosomal escape or replication and is mediated by a heat-stable and constitutively expressed bacterial factor. An analysis of the molecular mechanism of STAT1 inhibition indicated that expression of SOCS3, an established negative regulator of IFNgamma signaling, is highly up-regulated during infection and suppresses STAT1 phosphorylation. Functional analyses revealed that this interference with IFNgamma signaling is accompanied by the suppression of IP-10 production and iNOS induction resulting in increased intracellular bacterial survival. Importantly, the suppressive effect on IFNgamma-mediated host cell protection is most effective when IFNgamma is added post infection, suggesting that the bacteria establish a permissive environment within the host cell.

The pulmonary innate immune system responds to various airborne microbes. Although its specificity is broad and based on the recognition of pathogen-associated molecular patterns, it is uniquely regulated to limit inflammation and thereby prevent damage to the gas-exchanging alveoli. Macrophages, critical cell determinants of this system, recognize microbes through pattern recognition receptors such as TLRs, which typically mediate proinflammatory responses. The lung collectin, surfactant protein A (SP-A), has emerged as an important innate immune determinant that regulates microbe-macrophage interactions in this environment. In this study, we report the basal and SP-A-induced transcriptional and posttranslational regulation of TLR2 and TLR4 expression during the differentiation of primary human monocytes into macrophages. Despite SP-A's ability to up-regulate TLR2 expression on human macrophages, it dampens TLR2 and TLR4 signaling in these cells. SP-A decreases the phosphorylation of IkappaBalpha, a key regulator of NF-kappaB activity, and nuclear translocation of p65 which result in diminished TNF-alpha secretion in response to TLR ligands. SP-A also reduces the phosphorylation of TLR signaling proteins upstream of NF-kappaB, including members of the MAPK family. Finally, we report for the first time that SP-A decreases the phosphorylation of Akt, a major cell regulator of NF-kappaB and potentially MAPKs. These data identify a critical role for SP-A in modulating the lung inflammatory response by regulating macrophage TLR activity.

We describe the first "Mycobacterium paraffinicum" (unofficial taxon) pseudo-outbreak in a tertiary-care medical center. Fifteen clinical nontuberculous mycobacterium isolates from 10 patients were initially identified by biochemical tests and high-performance liquid chromatography as Mycobacterium scrofulaceum. However, further testing by molecular analysis revealed "M. paraffinicum." Epidemiological and environmental investigation determined that the ice machine was the source of the pseudo-outbreak.

Worldwide clinical cases due to multi drug- and extensively drug-resistant strains of Mycobacterium tuberculosis (M.tb) are increasing making the need for new therapies more critical than ever. A major obstacle for designing new drugs to treat mycobacterial infections is our limited knowledge of the interface between the bacillus (especially M.tb) and its host. The pulmonary innate immune system plays a key role in the recognition of microbes entering via the respiratory route. Although the specificity of this system is broad and based on the recognition of pathogen-associated molecular patterns (PAMPs), it is uniquely regulated to limit inflammation and thereby prevent damage to the gas-exchanging alveoli. Pulmonary surfactant proteins A and D (SP-A and SP-D) are collagenous, soluble, C-type (Ca(2+)-dependent) lectins (named collectins) of the lung innate immune system that are secreted into the alveoli by resident type II alveolar epithelial cells and distal bronchiolar Clara cells. The related collectin in serum, mannose-binding lectin/protein (MBL or MBP), provides first-line defense against several microbes. Phagocytes represent the first cellular defense in the alveoli and their surface is rich in C-type lectin pattern recognition receptors (PRRs), including the mannose receptor (MR), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) and DC-associated C-type lectin-1 (Dectin-1). This review will discuss the important roles of the cell-associated C-type lectin PRRs and soluble collectins in the innate immune response to mycobacterial infections, and will present the current state of knowledge regarding the potential uses of these C-type lectins in therapy against infections, focusing on M.tb.

Iron (Fe) acquisition is essential for the growth of intracellular Mycobacterium tuberculosis (M.tb). How this occurs is poorly understood. Hereditary hemochromatosis is an inherited disease in which most cells become overloaded with Fe. However, hereditary hemochromatosis macrophages have lower than normal levels of intracellular Fe. This suggests M.tb growth should be slower in those cells if macrophage intracellular Fe is used by M.tb. Therefore, we compared trafficking and acquisition of transferrin (Tf)- and lactoferrin (Lf)-chelated Fe by M.tb within the phagosome of monocyte-derived macrophages (MDM) from healthy controls and subjects with hereditary hemochromatosis. M.tb in both sets of macrophages acquired more Fe from Lf than Tf. Fe acquisition by M.tb within hereditary hemochromatosis macrophages was decreased by 84% from Tf and 92% from Lf relative to that in healthy control macrophages. There was no difference in Fe acquired from Tf and Lf by the two macrophage phenotypes. Both acquired 3 times more Fe from Lf than Tf. M.tb infection and incubation with interferon gamma (IFN-gamma) reduced macrophage Fe acquisition by 20% and 50%, respectively. Both Tf and Lf colocalized with M.tb phagosomes to a similar extent, independent of macrophage phenotype. M.tb growth was 50% less in hereditary hemochromatosis macrophages. M.tb growing within macrophages from subjects with hereditary hemochromatosis acquire less Fe compared with healthy controls. This is associated with reduced growth of M.tb. These data support a role for macrophage intracellular Fe as a source for M.tb growth.

Hepcidin is an antimicrobial peptide produced by the liver in response to inflammatory stimuli and iron overload. Hepcidin regulates iron homeostasis by mediating the degradation of the iron export protein ferroportin 1, thereby inhibiting iron absorption from the small intestine and release of iron from macrophages. Here, we examined the expression of hepcidin in macrophages infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis. Stimulation of the mouse RAW264.7 macrophage cell line and mouse bone marrow-derived macrophages with mycobacteria and IFN-gamma synergistically induced high levels of hepcidin mRNA and protein. Similar results were obtained using the human THP-1 monocytic cell line. Stimulation of macrophages with the inflammatory cytokines IL-6 and IL-beta did not induce hepcidin mRNA expression. Iron loading inhibited hepcidin mRNA expression induced by IFN-gamma and M. avium, and iron chelation increased hepcidin mRNA expression. Intracellular protein levels and secretion of hepcidin were determined by a competitive chemiluminescence ELISA. Stimulation of RAW264.7 cells with IFN-gamma and M. tuberculosis induced intracellular expression and secretion of hepcidin. Furthermore, confocal microscopy analyses showed that hepcidin localized to the mycobacteria-containing phagosomes. As hepcidin has been shown to possess direct antimicrobial activity, we investigated its activity against M. tuberculosis. We found that hepcidin inhibited M. tuberculosis growth in vitro and caused structural damage to the mycobacteria. In summary, our data show for the first time that hepcidin localizes to the phagosome of infected, IFN-gamma-activated cells and has antimycobacterial activity.

AcpA of Francisella spp. is a respiratory-burst-inhibiting acid phosphatase that also exhibits phospholipase C activity. To better understand the molecular basis of AcpA in virulence, a deletion of acpA was constructed in Francisella novicida. The phosphatase and lipase activities were reduced 10-fold and 8-fold, respectively, in the acpA mutant compared to the wild type and were found mostly associated with the outer membrane. The acpA mutant was more susceptible to intracellular killing than the wild-type strain in the THP-1 human macrophage-like cell line. In addition, mice infected with the acpA mutant survived longer than the wild-type strain and were less fit than the wild-type strain in competition infection assays. Transmission electron microscopy showed that the acpA mutant was delayed in escape from macrophage phagosomes, as more than 75% of acpA mutant bacteria could still be found inside phagosomes after 12 h of infection in THP-1 cells and human monocyte-derived macrophages, whereas most of the wild-type bacteria had escaped from the phagosome by 6 h postinfection. Thus, AcpA affects intracellular trafficking and the fate of Francisella within host macrophages.