Publications

The inorganic polyphosphate (poly-P) is a key regulator of stress responses and virulence in many bacterial pathogens including Campylobacter jejuni. The role of exopolyphosphatases/guanosine pentaphosphate (pppGpp) phosphohydrolases (PPX/GPPA) in poly-P homeostasis and C. jejuni pathobiology remains unexplored. Here, we analyzed deletion mutants (∆ppx1, ∆ppx2) and the double knockout mutant (dkppx), all ∆ppx mutants exhibited increased capacity to accumulate poly-P; however only ∆ppx1 and dkppx mutants showed decreased accumulation of ppGpp, an alarmone molecule that regulates stringent response in bacteria, suggesting potential dual role for PPX1/GPPA. Nutrient survival defect of ∆ppx mutants was rescued by the supplementation of specific amino acids implying that survival defect may be associated with decreased ppGpp and/ or increased poly-P in ∆ppx mutants. The ppk1 and spoT were upregulated in both ∆ppx1 and ∆ppx2 suggesting a compensatory role for SpoT and Ppk1 in poly-P and ppGpp homeostasis. The lack of ppx genes resulted in defects in motility, biofilm formation, nutrient stress survival, invasion and intracellular survival indicating that maintaining a certain level of poly-P is critical for ppx genes in C. jejuni pathophysiology. Both ppx1 and ppx2 mutants were resistant to human complement-mediated killing; however, the dkppx mutant was sensitive. The serum susceptibility did not occur in the presence of MgCl 2 and EGTA suggesting an involvement of the classical or lectin pathway of complement mediated killing. Interestingly, the chicken serum did not have any effect on the ∆ppx mutants' survival. The observed serum susceptibility was not related to C. jejuni surface capsule and lipooligosaccharide structures. Our study underscores the importance of PPX/GPPA proteins in poly-P and ppGpp homeostasis, two critical molecules that modulate environmental stress responses and virulence in C. jejuni.

Systemic inflammation that occurs with increasing age (inflammaging) is thought to contribute to the increased susceptibility of the elderly to several disease states. The elderly are at significant risk for developing pulmonary disorders and infectious diseases, but the contribution of inflammation in the pulmonary environment has received little attention. In this study, we demonstrate that the lungs of old mice have elevated levels of proinflammatory cytokines and a resident population of highly activated pulmonary macrophages that are refractory to further activation by IFN-γ. The impact of this inflammatory state on macrophage function was determined in vitro in response to infection with M.tb. Macrophages from the lungs of old mice secreted more proinflammatory cytokines in response to M.tb infection than similar cells from young mice and also demonstrated enhanced M.tb uptake and P-L fusion. Supplementation of mouse chow with the NSAID ibuprofen led to a reversal of lung and macrophage inflammatory signatures. These data indicate that the pulmonary environment becomes inflammatory with increasing age and that this inflammatory environment can be reversed with ibuprofen.

One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.

Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.

Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.

Mannosylated molecules on the Mycobacterium tuberculosis surface are important determinants in the immunopathogenesis of tuberculosis. To date, much attention has been paid to mannose-capped lipoarabinomannan, which mediates phagocytosis and intracellular trafficking of M. tuberculosis by engaging the macrophage mannose receptor and subsequently binds to intracellular CD1b molecules for presentation to T cells. Another important mannosylated lipoglycan on the M. tuberculosis surface is lipomannan (LM). Comparative structural detail of the LMs from virulent and avirulent strains is limited as is knowledge regarding their differential capacity to be recognized by the adaptive immune response. Here, we purified LM from the avirulent M. smegmatis and the virulent M. tuberculosis H(37)R(v), performed a comparative structural biochemical analysis, and addressed their ability to stimulate CD1b-restricted T cell clones. We found that M. tuberculosis H(37)R(v) produces a large neutral LM (TB-LM); in contrast, M. smegmatis produces a smaller linear acidic LM (SmegLM) with a high succinate content. Correspondingly, TB-LM was not as efficiently presented to CD1b-restricted T cells as SmegLM. Thus, here we correlate the structure-function relationships for LMs with CD1b-restricted T cell responses and provide evidence that the structural features of TB-LM contribute to its diminished T cell responsiveness.

Tuberculosis is still a major health problem in the world. Initial interactions between Mycobacterium tuberculosis and the host mark the pathway of infection and the subsequent host inflammatory response. This inflammatory response is tightly regulated by both the host and the bacterium during different stages of infection. As infection progresses, the initial intense pro-inflammatory response observed is regulated by suppressive mediators balancing inflammation. In this environment, M. tuberculosis battles to survive interfering with the host inflammatory response. In this review we discuss the major effector molecules involved in inflammation in relation to the different stages of M. tuberculosis infection.

Reactive oxygen species (ROS) are produced predominantly by phagocytic cells in response to microbial infections. When produced at optimal levels ROS have potent antimicrobial properties. However, excessive production of ROS induces apoptosis/necrosis of infected as well as bystander cells, resulting in inflammatory pathology. Previously we showed that vaccination of pigs with a modified live porcine reproductive and respiratory syndrome virus vaccine (PRRS-MLV) administered intranasally with a potent mucosal adjuvant M. tuberculosis whole-cell lysate (Mtb WCL) induces protective immunity against PRRSV challenge. In this study, using bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells harvested from that study were quantified for the levels of ROS produced. Our results indicated that in vaccinated pigs, levels of ROS were lower compared to unvaccinated PRRSV-challenged pigs. In unvaccinated but PRRSV-challenged pigs, the higher ROS production was associated with increased inflammatory lung pathology. In conclusion, our results suggest that intranasal immunization using PRRS-MLV along with a potent mucosal adjuvant protects pigs against both homologous and virulent heterologous PRRSV challenge, which was associated with reduced ROS production and reduced lung pathology compared to control virus-challenged pigs.