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Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species (ROS) due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by PMN of CGD patients (CGD PMN) and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in PMN from healthy subjects (normal PMN) and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of antiapoptotic genes (e.g., XIAP and GADD45B) and downregulation of proapoptotic genes (e.g., CASP8 and APAF1) in infected PMN. Transcript and protein levels of inflammation- and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered the expression of ROS resistance genes in the presence of normal but not CGD PMN. Levels of bacterial stress response genes, including the ClpB gene, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knockdown demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included the upregulation of pyruvate dehydrogenase. Pharmacological inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis of Granulibacter in cells from permissive (CGD) and nonpermissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

Invasive aspergillosis is a major threat for patients suffering from chronic granulomatous disease (CGD). Although Aspergillus fumigatus is the most commonly encountered Aspergillus species, the presence of A. nidulans appears to be disproportionately high in CGD patients. The purpose of this study was to investigate the involvement of the NADPH oxidase and the resulting reactive oxygen species (ROS) in host defense against fungi and to clarify their relationship toward A. nidulans. Murine CGD alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from healthy controls and CGD patients were challenged with either A. fumigatus or A. nidulans. Analysis of the antifungal effects of ROS revealed that A. nidulans, in contrast to A. fumigatus, is not susceptible to ROS. In addition, infection with live A. nidulans did not result in any measurable ROS release. Remarkably, human CGD PMN and PBMC and murine CGD AM were at least equipotent at arresting conidial germination compared to healthy controls. Blocking of the NADPH oxidase resulted in significantly reduced damage of A. fumigatus but did not affect A. nidulans hyphae. Furthermore, the microbicidal activity of CGD PMN was maintained toward A. nidulans but not A. fumigatus. In summary, antifungal resistance to A. nidulans is not directly ROS related. The etiology of A. nidulans infections in CGD cannot be explained by the simple absence of the direct microbicidal effect of ROS. In vivo, the NADPH oxidase is a critical regulator of innate immunity whose unraveling will improve our understanding of fungal pathogenesis in CGD.

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1beta. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.

Urinary nitrite and F(2)-isoprostanes, an index of oxidant stress, were elevated during chlamydial genital infection of mice. Enhancement of urinary nitrite and F(2)-isoprostanes was observed in phagocyte oxidase-deficient mice. Inhibition of inducible nitric oxide synthase reduced isoprostane excretion. We conclude that nitrogen radicals induce F(2)-isoprostane production and excretion during murine chlamydial genital infection.

Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens. Using mice deficient in generating phagocyte superoxide (p47(phox)(-/-)) and mice deficient in generating inducible nitric oxide synthase (iNOS(-/-)), we examined the roles of these reactive species in host defense against Burkholderia cepacia and Chromobacterium violaceum, organisms known to have unusual virulence in chronic granulomatous disease. Intraperitoneal B. cepacia challenge (4.0 x 10(3) to 4.0 x 10(5) organisms/mouse) resulted in mortality in all p47(phox)(-/-) mice, with the survival interval being inversely proportionate to the amount of inoculum. Pretreatment with gamma interferon did not affect survival. C. violaceum was strikingly virulent in p47(phox)(-/-) mice (the 50% lethal dose [LD(50)] was <13 organisms). iNOS(-/-) and wild-type mice were resistant to B. cepacia challenges of at least 10(6) organisms per mouse, and the LD(50) of C. violaceum was between 10(6) and 10(7) organisms per mouse. Consistent with the survival data, numbers of organisms in cultures of B. cepacia from multiple sites were higher for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice at day 4 after challenge, but numbers of organisms for different B. cepacia strains varied. The recovery of C. violaceum was strikingly greater at 18 h after challenge for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice, in which the organism burdens were virtually nil. In vitro, both B. cepacia and C. violaceum were sensitive to H(2)O(2) and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly. Host defense against B. cepacia and C. violaceum is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.

Salmonella enterica serovar Typhimurium is a gram-negative, facultative intracellular pathogen that predominantly invades mononuclear phagocytes and is able to establish persistent infections. One of the innate defense mechanisms of phagocytic cells is the production of reactive oxygen species, including superoxide. S. enterica serovar Typhimurium has evolved mechanisms to resist such radicals, and these mechanisms could be decisive in its ability to survive and replicate within macrophages. Recently, we described a superoxide-hypersusceptible S. enterica serovar Typhimurium mutant strain, DLG294, that carries a transposon in sspJ, resulting in the lack of expression of SspJ, which is necessary for resistance against superoxide and replication within macrophages. Here we show that DLG294, which is a 14028s derivative, hardly induced any granulomatous lesions in the livers upon subcutaneous infection of C3H/HeN (Ity(r)) mice with 3 x 10(4) bacteria and that its bacterial counts were reduced by 3 log units compared to those of wild-type S. enterica serovar Typhimurium 14028s on day 5 after infection. In contrast, DLG294 replicated like wild-type S. enterica serovar Typhimurium 14028s and induced a phenotypically similar liver pathology in p47(phox-/-) mice, which are deficient in the p47(phox) subunit of the NADPH oxidase complex and which do not produce superoxide. Consistent with these results, DLG294 reached bacterial counts identical to those of wild-type S. enterica serovar Typhimurium 14028s in bone marrow-derived macrophages from p47(phox-/-) mice and in X-CGD PLB-985 cells at 24 h after challenge. These results indicate that SspJ plays a role in the bacterium's resistance to oxidative stress and in the survival and replication of S. enterica serovar Typhimurium both in vitro and in vivo.

It has been previously reported that although inducible nitric oxide synthase (iNOS) gene knockout (NOS2(-/-)) mice resolve Chlamydia trachomatis genital infection, the production of reactive nitrogen species (RNS) via iNOS protects a significant proportion of mice from hydrosalpinx formation and infertility. We now report that higher in vivo RNS production correlates with mouse strain-related innate resistance to hydrosalpinx formation. We also show that mice with a deletion of a key component of phagocyte NADPH oxidase (p47(phox-/-)) resolve infection, produce greater amounts of RNS in vivo, and sustain lower rates of hydrosalpinx formation than both wild-type (WT) NOS2(+/+) and NOS2(-/-) controls. When we induced an in vivo chemical block in iNOS activity in p47(phox-/-) mice using N(G)-monomethyl-L-arginine (L-NMMA), a large proportion of these mice eventually succumbed to opportunistic infections, but not before they resolved their chlamydial infections. Interestingly, when compared to WT and untreated p47(phox-/-) controls, L-NMMA-treated p47(phox-/-) mice resolved their infections more rapidly. However, L-NMMA-treated p47(phox-/-) mice lost resistance to chronic chlamydial disease, as evidenced by an increased rate of hydrosalpinx formation that was comparable to that for NOS2(-/-) mice. We conclude that phagocyte oxidase-derived reactive oxygen species (ROS) regulate RNS during chlamydial urogenital infection in the mouse. We further conclude that while neither phagocyte oxidase-derived ROS nor iNOS-derived RNS are essential for resolution of infection, RNS protect from chronic chlamydial disease in this model.

Reactive oxygen species (ROS) are thought to be involved in intracellular signaling, including activation of the transcription factor NF-kappaB. We investigated the role of NADPH oxidase in the NF-kappaB activation pathway by utilizing knockout mice (p47phox-/-) lacking the p47phox component of NADPH oxidase. Wild-type (WT) controls and p47phox-/- mice were treated with intraperitoneal (i.p.) Escherichia coli lipopolysaccharide (LPS) (5 or 20 microg/g of body weight). LPS-induced NF-kappaB binding activity and accumulation of RelA in nuclear protein extracts of lung tissue were markedly increased in WT compared to p47phox-/- mice 90 min after treatment with 20 but not 5 microg of i.p. LPS per g. In another model of lung inflammation, RelA nuclear translocation was reduced in p47phox-/- mice compared to WT mice following treatment with aerosolized LPS. In contrast to NF-kappaB activation in p47phox-/- mice, LPS-induced production of macrophage inflammatory protein 2 in the lungs and neutrophilic lung inflammation were not diminished in these mice compared to WT mice. We conclude that LPS-induced NF-kappaB activation is deficient in the lungs of p47phox-/- mice compared to WT mice, but this abnormality does not result in overt alteration in the acute inflammatory response.

Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide and downstream microbicidal reactive oxidants, leading to recurrent life-threatening bacterial and fungal infections. Xanthine oxidase (XO) is another enzyme known to produce superoxide in many tissues. Using the p47(phox-/-) mouse model of CGD, we evaluated the residual antibacterial activity of XO. Clearance of Burkholderia cepacia, a major pathogen in CGD, was reduced in p47(phox-/-) mice compared to that in wild-type mice and was further inhibited in p47(phox-/-) mice by pretreatment with the specific XO inhibitor allopurinol. Hepatic B. cepacia burden was similar in the two genotypes, but allopurinol significantly reduced net hepatic killing and killing efficiency only in p47(phox-/-) mice. Clearance and killing of intravenous Escherichia coli was intact in p47(phox-/-) mice and was unaffected by pretreatment with allopurinol. In CGD, XO may contribute to host defense against a subset of reactive oxidant-sensitive pathogens.

Mycobacterium tuberculosis is an important respiratory pathogen the growth of which is controlled primarily by cytokine-activated macrophages. One of the principal mediators of this control is nitric oxide; however, superoxide has recently been shown to be protective in systemic mycobacterial infections. To determine whether superoxide is important in controlling M. tuberculosis during primary pulmonary infection, mice lacking the cytosolic p47(phox) gene (which is essential for effective superoxide production by the NADPH oxidase) were infected aerogenically. The lack of superoxide during an aerosol infection with M. tuberculosis resulted in a significant increase in bacterial growth over the early period of infection. Once antigen-specific gamma interferon-producing lymphocytes were detected in the draining lymph nodes, however, bacterial growth in the lung stopped. One interesting consequence of the lack of superoxide was an increase in neutrophilic infiltrates within the granuloma. This may be a consequence of increased tissue damage due to more rapid bacterial growth or may reflect a role for superoxide in controlling inflammation.

Chronic granulomatous disease (CGD) is a genetic disorder of NADPH oxidase in which phagocytes are defective in generating reactive oxidants. CGD patients suffer from recurrent infections and exuberant and persistent tissue granuloma formation. We hypothesized that abnormal granulomata in CGD may result from aberrant T-cell-mediated cytokine responses. To assess Th-1-type cytokine responses and granulomata, we challenged p47(phox-/-) and wild-type mice with avirulent (SmD) or virulent (SmT) variants of Mycobacterium avium 2-151. To assess Th-2-type cytokine responses and granulomata, we used Schistosoma mansoni eggs (SME). Mononuclear cells were harvested, and cytokine responses were determined by enzyme-linked immunosorbent assay or reverse transcriptase PCR. Following SmD or SmT challenge, splenocytes from p47(phox-/-) and wild-type mice generated similar polar Th-1 responses (increased levels of gamma interferon and basal levels of interleukin 4 [IL-4] and IL-5). By 8 weeks after SmT challenge, exuberant splenic granulomata developed in p47(phox-/-) and wild-type mice. After SME challenge, thoracic lymph node mononuclear cells from p47(phox-/-) and wild-type mice generated similar mixed Th-1 and Th-2 cytokine responses to SME antigen and concanavalin A. Peak lung granuloma sizes and rates of regression were similar in p47(phox-/-) and wild-type mice. These results suggest that exuberant granulomatous inflammation in CGD is probably not the result of skewing of T-cell responses toward the Th-1 or Th-2 pole. Appropriate regression of established tissue granulomata in p47(phox-/-) mice challenged with SME suggests that abnormal granuloma formation in CGD is stimulus dependent and is not an invariant feature of the disease.

Trachoma is a common blinding disease of humans caused by ocular infections with Chlamydia trachomatis. The cynomolgus monkey is a valuable primate model for the detection, pathobiology, and treatment of this infection. We have used this model system to compare the relative ability of tissue culture, direct fluorescence cytology, a modified polymerase chain reaction, and RNA blotting to detect C. trachomatis following primary infection and reinfection over 34 weeks. Six cynomolgus monkeys were given a primary ocular chlamydia infection, and 20 weeks later they were reinoculated with the same organism. All animals showed brisk inflammatory responses to the primary infection and milder inflammatory reactions to reinfection. All four diagnostic techniques detected chlamydia at 1 week after primary infection, but both nucleic acid detection methods suggested that organisms were present longer after primary infection than did either tissue culture or direct fluorescence cytology (16 weeks for RNA blotting versus 12 weeks for tissue culture). Following reinoculation at 20 weeks, the period of C. trachomatis detection by tissue culture or direct fluorescence cytology (4 weeks) was much shorter than after primary infection. In contrast, nucleic acid detection was positive for up to 5 weeks longer than tissue culture or direct fluorescence cytology. Both polymerase chain reaction and RNA blotting, which involved no amplification step, indicated the presence of organisms during the culture-negative period. These data suggest that live chlamydiae may remain at a site of infection and produce inflammation beyond the time at which standard microbiological techniques are able to detect them.

To serially examine the immunopathogenesis and histopathology of infection with Chlamydia pneumoniae, we inoculated two cynomolgus monkeys in the conjunctival sac, nose, and nasopharynx with C. pneumoniae TWAR. After inoculation, C. pneumoniae was isolated from the inoculation sites and the rectums of both monkeys for a period of 5 weeks. After a second inoculation, C. pneumoniae was recovered from the inoculation sites and the rectums of both monkeys for 20 weeks. A third inoculation with C. pneumoniae caused very little productive infection at any site. Prior C. pneumoniae infection did not prevent subsequent C. trachomatis serovar E (Bour strain) infection. Clinical and histopathologic ocular responses to C. pneumoniae infection were mild compared with those to infection with C. trachomatis serovar E. Rectal infection, demonstrated by culture isolation and immunohistopathology, occurred without direct experimental inoculation. Both immunofluorescent staining of mucosal smears with monoclonal antibodies and tissue culture were able to detect C. pneumoniae infection. Experimental nonhuman primate infection with C. pneumoniae appears to be clinically and histopathologically mild and can occur at extrapulmonary sites.