Androgen sensitivity gateway to COVID-19 disease severity.

In this communication, we present arguments for androgen sensitivity as a likely determinant of COVID-19 disease severity. The androgen sensitivity model explains why males are more likely to develop severe symptoms while children are ostensibly resistant to infection. Further, the model explains the difference in COVID-19 mortality rates among different ethnicities. Androgen sensitivity is determined by genetic variants of the androgen receptor. The androgen receptor regulates transcription of the transmembrane protease, serine 2 (TMPRSS2), which is required for SARS-CoV-2 infectivity. TMPRSS2 primes the Spike protein of the virus, which has two consequences: diminishing viral recognition by neutralizing antibodies and activating SARS-CoV-2 for virus-cell fusion. Genetic variants that have been associated with androgenetic alopecia, prostate cancer, benign prostatic hyperplasia and polycystic ovary syndrome could be associated with host susceptibility. In addition to theoretical epidemiological and molecular mechanisms, there are reports of high rates of androgenetic alopecia of from hospitalized COVID-19 patients due to severe symptoms. Androgen sensitivity is a likely determinant of COVID-19 disease severity. We believe that the evidence presented in this communication warrants the initiation of trials using anti-androgen agents.

A predicted Francisella tularensis DXD-motif glycosyltransferase blocks immune activation.

Pathogens enhance their survival during infections by manipulating host defenses. Francisella tularensis evades innate immune responses, which we have found to be dependent on an understudied gene ybeX (FTL_0883/FTT_0615c). To understand the function of YbeX, we sought protein interactors in F. tularensis subsp. holarctica live vaccine strain (LVS). An unstudied Francisella protein co-immunoprecipitated with recombinant YbeX, which is a predicted glycosyltransferase with a DXD-motif. There are up to four genomic copies of this gene with identical sequence in strains of F. tularensis pathogenic to humans, despite ongoing genome decay. Disruption mutations were generated by intron insertion into all three copies of this glycosyltransferase domain containing gene in LVS, gdcA1-3. The resulting strains stimulated more cytokines from macrophages in vitro than wild-type LVS and were attenuated in two in vivo infection models. GdcA was released from LVS during culture and was sufficient to block NF-κB activation when expressed in eukaryotic cells. When co-expressed in zebrafish, GdcA and YbeX were synergistically lethal to embryo development. Glycosyltransferases with DXD-motifs are found in a variety of pathogens including NleB, an Escherichia coli type-III secretion system effector that inhibits NF-κB by antagonizing death receptor signaling. To our knowledge, GdcA is the first DXD-motif glycosyltransferase that inhibits NF-κB in immune cells. Together, these findings suggest DXD-motif glycosyltransferases may be a conserved virulence mechanism used by pathogenic bacteria to remodel host defenses.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

The use of resazurin as a novel antimicrobial agent against Francisella tularensis.

The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 µM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 µM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. Resorufin also suppressed the growth of F. tularensis suggesting that this compound is the biologically active form responsible for decreasing the viability of F. tularensis LVS bacteria. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 µM resazurin indicating this compound could be an effective therapy for tularemia in vivo.

Role of NK cells in host defense against pulmonary type A Francisella tularensis infection.

Pneumonic tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Understanding the pulmonary immune response to this bacterium is necessary for developing effective vaccines and therapeutics. In this study, characterization of immune cell populations in the lungs of mice infected with the type A strain Schu S4 revealed a significant loss in natural killer (NK) cells over time. Since this decline in NK cells correlated with morbidity and mortality, we hypothesized these cells contribute to host defense against Schu S4 infection. Depletion of NK cells prior to Schu S4 challenge significantly reduced IFN-γ and granzyme B in the lung but had no effect on bacterial burden or disease progression. Conversely, increasing NK cell numbers with the anti-apoptotic cytokine IL-15 and soluble receptor IL-15Rα had no significant impact on Schu S4 growth in vivo. A modest decrease in median time to death, however, was observed in live vaccine strain (LVS)-vaccinated mice depleted of NK1.1+ cells and challenged with Schu S4. Therefore, NK cells do not appear to contribute to host defense against acute respiratory infection with type A F. tularensis in vivo, but they play a minor role in protection elicited by LVS vaccination.

Interferon-γ, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis (MTB) remains a leading infectious threat to human health. Macrophages are the cells targeted for infection by the bacterium as well as key effector cells for clearance of the pathogen. Interleukin (IL)-27 opposes macrophage-mediated control of MTB because supplying IL-12 and blocking the activity of IL-27 limits bacterial growth in primary human macrophages. The purpose of this study was to determine the immunological regulators of this macrophage mechanism to restrict MTB growth. Interferon (IFN)-γ, TNF-α, and IL-18 were all demonstrated to be important to the environment that limits bacterial growth when IL-12 is supplied and IL-27 is neutralized. We find IL-18 works in conjunction with IL-12 to achieve optimal IFN-γ production in this system. We also demonstrate novel interactions between these cytokines to influence the expression or responsiveness to one another. Quantitative assays show that IFN-γ enhances expression of the IL-18 receptor signaling chain, as well as TNF expression and secretion. In turn, TNF-α augments expression of the receptor for IFN-γ, the amount at the cell surface, and the extent of IFN-γ -induced signaling. We further define how the cytokine environment supports an enhanced state of classical macrophage activation. Collectively, these results describe novel immunological mechanisms that provide additional insights into the effects of IL-12 and IL-27 on macrophage regulation during MTB infection.

A Francisella tularensis live vaccine strain that improves stimulation of antigen-presenting cells does not enhance vaccine efficacy.

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence.

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.

Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Cytokines involved in interferon-gamma production by human macrophages.

Interferon (IFN)-gamma is important to the immune defense against intracellular pathogens and specifically the ability of macrophages to control Mycobacterium tuberculosis (MTB). Increasing evidence has accumulated to support the idea that macrophages produce IFN-gamma. We describe here the cytokine interactions that determine IFN-gamma expression and secretion during MTB infection of human macrophages. Detection of biologically important IFN-gamma levels in culture supernatants of MTB-infected human macrophages requires the addition of interleukin (IL)-12. IL-18 augmented IFN-gamma production from human macrophages in response to the combination of MTB and supplemental IL-12. Although IL-18 gene expression was generally unchanged, IL-18 protein secretion was enhanced by the combination of MTB and IL-12, and functioned primarily to stimulate IFN-gamma release. Importantly, IL-27 induced by MTB infection opposed IFN-gamma production by antagonizing IL-18 activity in human macrophages. Neutralization of IL-27 increased the expression of the IL-18 receptor beta-chain. Additionally, IL-27 blocked NF-kappaB activation in response to IL-18. These results define the signals required for IFN-gamma production by human macrophages and highlight the interactions between cytokines produced during MTB infection. Together, they identify a novel role for IL-27 in regulating macrophage function by disrupting IL-18 activity.

Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo.

The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized DeltapyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis DeltapyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis DeltapyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis DeltapyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis DeltapyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

Cross species expression analysis of innate immune response.

The innate immune response is the first line of host defense against infections. This system employs a number of different types of cells, which in turn activate different sets of genes. Microarray studies of human and mouse cells infected with various pathogens identified hundreds of differentially expressed genes. However, combining these datasets to identify common and unique response patterns remained a challenge. We developed methods based on probabilistic graphical models to combine expression experiments across species, cells, and pathogens. Our method analyzes homologous genes in different species concurrently overcoming problems related to noise and orthology assignments. Using our method, we identified both core immune response genes and genes that are activated in macrophages in both human and mouse but not in dendritic cells, and vice versa. Our results shed light on immune response mechanisms and on the differences between various types of cells that are used to fight infecting bacteria. For supporting website, see www.cs.cmu.edu/-lyongu/pub/immune/.

Global transcriptional response to spermine, a component of the intramacrophage environment, reveals regulation of Francisella gene expression through insertion sequence elements.

Tularemia is caused by the category A biodefense agent Francisella tularensis. This bacterium is associated with diverse environments and a plethora of arthropod and mammalian hosts. How F. tularensis adapts to these different conditions, particularly the eukaryotic intracellular environment in which it replicates, is poorly understood. Here, we demonstrate that the polyamines spermine and spermidine are environmental signals that alter bacterial stimulation of host cells. Genomewide analysis showed that F. tularensis LVS undergoes considerable changes in gene expression in response to spermine. Unexpectedly, analysis of gene expression showed that multiple members of two classes of Francisella insertion sequence (IS) elements, ISFtu1 and ISFtu2, and the genes adjacent to these elements were induced by spermine. Spermine was sufficient to activate transcription of these IS elements and of nearby genes in broth culture and in macrophages. Importantly, the virulent strain of F. tularensis, Schu S4, exhibited similar phenotypes of cytokine induction and gene regulation in response to spermine. Distinctions in gene expression changes between Schu S4 and LVS at one orthologous locus, however, correlated with differences in IS element location. Our results indicate that spermine and spermidine are novel triggers to alert F. tularensis of its eukaryotic host environment. The results reported here also identify an unexpected mechanism of gene regulation controlled by a spermine-responsive promoter contained within IS elements. Different arrangements of these mobile genetic elements among Francisella strains may contribute to virulence by conveying new expression patterns for genes from different strains.

Global transcriptional response to mammalian temperature provides new insight into Francisella tularensis pathogenesis.

BACKGROUND: After infecting a mammalian host, the facultative intracellular bacterium, Francisella tularensis, encounters an elevated environmental temperature. We hypothesized that this temperature change may regulate genes essential for infection. RESULTS: Microarray analysis of F. tularensis LVS shifted from 26 degrees C (environmental) to 37 degrees C (mammalian) showed approximately 11% of this bacterium's genes were differentially-regulated. Importantly, 40% of the protein-coding genes that were induced at 37 degrees C have been previously implicated in virulence or intracellular growth of Francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. Forty-four percent of the genes induced at 37 degrees C encode proteins of unknown function, suggesting novel Francisella virulence traits are regulated by mammalian temperature. To explore this possibility, we generated two mutants of loci induced at 37 degrees C [FTL_1581 and FTL_1664 (deoB)]. The FTL_1581 mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. FTL_1581 encodes a novel hypothetical protein that we suggest naming temperature-induced, virulence-associated locus A, tivA. Interestingly, the deoB mutant showed diminished entry into mammalian cells compared to wild-type LVS, including primary human macrophages and dendritic cells, the macrophage-like RAW 264.7 line, and non-phagocytic HEK-293 cells. This is the first study identifying a Francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells. CONCLUSION: Our results provide new insight into mechanisms of Francisella virulence regulation and pathogenesis. F. tularensis LVS undergoes considerable gene expression changes in response to mammalian body temperature. This temperature shift is important for the regulation of genes that are critical for the pathogenesis of Francisella. Importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tivA (FTL_1581). An analysis of tivA and deoB (FTL_1664) revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively.

IL-1beta-driven ST2L expression promotes maturation resistance in rapamycin-conditioned dendritic cells.

Maturation resistance and tolerogenic properties can be conferred on human and murine dendritic cells (DC), crucial regulators of T cell responses, by exposure to rapamycin (RAPA), a "tolerance-sparing" immunosuppressive agent. Mechanisms underlying this acquired unresponsiveness, typified by diminished functional responses to TLR or CD40 ligation, have not been identified. We report that in vitro and in vivo conditioning of murine myeloid DC with RAPA elicits the de novo production of IL-1beta by otherwise phenotypically immature DC. Interestingly, IL-1beta production promotes overexpression of the transmembrane form of the IL-1R family member, IL-1R-like 1, also know as ST2 on RAPA-conditioned DC (RAPA-DC). ST2 is the recently identified receptor for IL-33, a cytokine favoring Th2 responses. In addition, transmembrane ST2, or ST2L, has been implicated as a potent negative regulator of TLR signaling. RAPA-DC generated from ST2-/- mice exhibited higher levels of costimulatory molecules (CD86) than wild-type RAPA-DC. Consistent with its regulatory function, IL-1beta-induced ST2L expression suppressed the responsiveness of RAPA-DC to TLR or CD40 ligation. Thus, as a result of their de novo production of IL-1beta, RAPA-DC up-regulate ST2L and become refractory to proinflammatory, maturation-inducing stimuli. This work identifies a novel mechanism through which a clinically important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.

Interleukin-12 and interleukin-27 regulate macrophage control of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is an intracellular pathogen that persists within macrophages and remains a considerable global threat to human health. The purpose of this study was to investigate how interleukin (IL)-12 and IL-27 regulate human macrophage interactions with M. tuberculosis. Quantitative measurement of transcripts showed that IL-12 or M. tuberculosis induced IL-27 gene expression in human macrophages. Furthermore, IL-27 receptor subunits were shown by reverse transcription-polymerase chain reaction and flow cytometry to be expressed and present at the cell surface. Neutralization of IL-27 in the presence of IL-12 reduced viable M. tuberculosis recovered from macrophages. Antimycobacterial activity was accompanied by a heightened inflammatory response that included tumor necrosis factor, IL-6, interferon-gamma, and a subset of chemokines. These results implicate IL-12 and IL-27 in regulating human macrophages, and IL-27 derived from macrophages during infection impedes control of M. tuberculosis growth.

The cyclic AMP-dependent catabolite repression system of Serratia marcescens mediates biofilm formation through regulation of type 1 fimbriae.

The mechanisms by which environmental carbon sources regulate biofilm formation are poorly understood. This study investigates the roles of glucose and the catabolite repression system in Serratia marcescens biofilm formation. The abilities of this opportunistic pathogen to proliferate in a wide range of environments, to cause disease, and to resist antimicrobials are linked to its ability to form biofilms. We observed that growth of S. marcescens in glucose-rich medium strongly stimulated biofilm formation, which contrasts with previous studies showing that biofilm formation is inhibited by glucose in Escherichia coli and other enteric bacteria. Glucose uptake is known to inversely mediate intracellular cyclic AMP (cAMP) synthesis through regulation of adenylate cyclase (cyaA) activity, which in turn controls fundamental processes such as motility, carbon utilization and storage, pathogenesis, and cell division in many bacteria. Here, we demonstrate that mutation of catabolite repression genes that regulate cAMP levels (crr and cyaA) or the ability to respond to cAMP (crp) confers a large increase in biofilm formation. Suppressor analysis revealed that phenotypes of a cAMP receptor protein (crp) mutant require the fimABCD operon, which is responsible for type 1 fimbria production. Consistently, fimA transcription and fimbria production were determined to be upregulated in a cyaA mutant background by using quantitative real-time reverse transcription-PCR and transmission electron microscopy analysis. The regulatory pathway by which environmental carbon sources influence cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development.

Idiopathic vertebral abscess in pregnancy: case report and literature review.

Pregnant women are prone to complications from many infections. However, overwhelming soft tissue infections are rarely reported in pregnant women without predisposing factors. A 40-year-old multipara presented with complaints of progressively worsening spinal muscle spasm pain. Because her medical history consisted of preterm deliveries, she was receiving injections of 17-alpha-hydroxyprogesterone caproate (17P). An imaging study revealed replacement of the vertebral marrow with a homogeneous substance. A biopsy stained for gram-positive cocci. Blood culture bottles grew STAPHYLOCOCCUS AUREUS. The lesion was re-aspirated, and grew the same isolate. The patient was treated with antibiotics and had resolution of her pain. Mother and infant did well, and she completed a prolonged course of antibiotics. This study presents an unusual case of idiopathic vertebral osteomyelitis and epidural abscess during pregnancy. Potential reasons for failure to display a systemic response to this infection with associated bacteremia include an innate variation in the patient's immune system and modification in immunity from progesterone therapy. The role that 17P played in this case, if any, is unknown.

Modulation of virulence factors in Francisella tularensis determines human macrophage responses.

Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis live vaccine strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a sigma(54)-modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically defined medium. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses.

Photoinactivation of Mycobacteria in vitro and in a new murine model of localized Mycobacterium bovis BCG-induced granulomatous infection.

Treatment of tuberculosis is currently hindered by prolonged antibiotic regimens and the emergence of significant drug resistance. Alternatives and adjuncts to standard antimycobacterial agents are needed. We propose that a direct attack utilizing photosensitizers and light-based treatments may be effective in curtailing Mycobacterium tuberculosis in discrete anatomical sites in the most infectious phase of pulmonary tuberculosis. To demonstrate experimental proof of principle, we have applied established photodynamic therapy (PDT) technology to in vitro cultures and an in vivo mouse model using Mycobacterium bovis BCG. We report here in vitro and in vivo PDT efficacy studies and the use of a three-dimensional collagen gel as a delivery vehicle for BCG, subcutaneously inserted, to induce specifically localized granuloma-like lesions in mice. When a benzoporphyrin derivative was utilized as the photosensitive agent, exposure to light killed extracellular and intracellular BCG in significant numbers. Collagen scaffolds containing BCG inserted in situ in BALB/c mice for 3 months mimicked granulomatous lesions and demonstrated a marked cellular infiltration upon histological examination, with evidence of caseating necrosis and fibrous capsule formation. When 10(5) BCG were present in the in vivo-induced granulomas, a significant reduction in viable mycobacterial cells was demonstrated in PDT-treated granulomas compared to those of controls. We conclude that PDT has potential in the treatment of localized mycobacterial infections, such as pulmonary granulomas and cavities.