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Bakaletz, “Biological roles of nontypeable Haemophilus influenzae type IV pilus proteins encoded by the pil and com operons
- Journal of Bacteriology
, 2012
"... We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeableHaemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD an ..."
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We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeableHaemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence de-fect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a bio-film in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling andmass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic. Nontypeable Haemophilus influenzae (NTHI) is a commensalof the human airway, yet it can cause multiple upper and lower respiratory tract diseases when host immune defenses are
Identification of Gene Products Involved in the Oxidative Stress Response of Moraxella catarrhalis†
, 2010
"... Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990–3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by ..."
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Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990–3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by exogenous hydrogen peroxide (H2O2). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated in M. catarrhalis strain O35E, resulting in an increase in sensitivity to killing by H2O2 in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H2O2. DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identified M. catarrhalis genes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing of M. catarrha-lis O35E katA and ahpC mutants for their abilities to scavenge exogenous H2O2 showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations into M. catarrhalis strain ETSU-4 showed that the growth of a ETSU-4 katA mutant was markedly inhibited by the addition of 50 mM H2O2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain. Moraxella catarrhalis is an aerobic, Gram-negative coccoba-
0021-9193/09/$12.00 doi:10.1128/JB.00619-09 Global Effects of Inactivation of the Pyruvate Kinase Gene in the Mycobacterium tuberculosis Complex†
, 2009
"... To better understand the global effects of “natural ” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis pykA mutant yield ..."
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To better understand the global effects of “natural ” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis pykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the pykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in -oxidation were increased in the M. tuberculosis pykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the pykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the pykA knockout, explaining why an M. tuberculosis pykA ald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has
Peroxide-Sensing Transcriptional Regulators in Bacteria
"... The ability to maintain intracellular concentrations of toxic reactive oxygen species (ROS) within safe limits is essential for all aerobic life forms. In bacteria, as well as other organisms, ROS are produced during the normal course of aerobic metabolism, necessitating the constitutive expression ..."
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The ability to maintain intracellular concentrations of toxic reactive oxygen species (ROS) within safe limits is essential for all aerobic life forms. In bacteria, as well as other organisms, ROS are produced during the normal course of aerobic metabolism, necessitating the constitutive expression of ROS scavenging systems. However, bacteria can also experience transient high-level exposure to ROS derived either from external sources, such as the host defense response, or as a secondary effect of other seem-ingly unrelated environmental stresses. Consequently, transcriptional regulators have evolved to sense the levels of ROS and coordinate the appropriate oxidative stress response. Three well-studied examples of these are the peroxide responsive regula-tors OxyR, PerR, and OhrR. OxyR and PerR are sensors of primarily H2O2, while OhrR senses organic peroxide (ROOH) and sodium hypochlorite (NaOCl). OxyR and OhrR sense oxidants by means of the reversible oxidation of specific cysteine residues. In contrast, PerR senses H2O2 via the Fe-catalyzed oxidation of histidine residues. These transcription regulators also influence complex biological phenomena, such as biofilm formation, the evasion of host immune responses, and antibiotic resistance via the direct regulation of specific proteins. An effective oxidative stress defense response is a required itemin the basic survival kit of all aerobic organisms as well as those anaerobes that exist in environments subject to transient exposures to oxygen. This is due to molecular oxygen’s ability to accept electrons from cellular redox components to form toxic
Global Effects of Inactivation of the Pyruvate Kinase Gene in the Mycobacterium tuberculosis Complex � †
, 2009
"... To better understand the global effects of “natural ” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis �pykA mutant yiel ..."
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To better understand the global effects of “natural ” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis �pykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the �pykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in �-oxidation were increased in the M. tuberculosis �pykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the �pykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the �pykA knockout, explaining why an M. tuberculosis �pykA �ald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has
Haemophilus influenzae OxyR: Characterization of Its Regulation, Regulon and Role in Fitness
"... To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulat ..."
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To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a
