Comparative study of the roles of AhpC and KatE as respiratory antioxidants in Brucella abortus 2308.

Brucella strains are exposed to potentially toxic levels of H2O2 both as a consequence of their aerobic metabolism and through the respiratory burst of host phagocytes. To evaluate the relative contributions of the sole catalase KatE and the peroxiredoxin AhpC produced by these strains in defense against H2O2-mediated toxicity, isogenic katE, ahpC, and katE ahpC mutants were constructed and the phenotypic properties of these mutants compared with those of the virulent parental strain B. abortus 2308. The results of these studies indicate that AhpC is the primary detoxifier of endogenous H2O2 generated by aerobic metabolism. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H2O2, although this enzyme can play a supporting role in the detoxification of H2O2 of endogenous origin if AhpC is absent. B. abortus ahpC and katE mutants exhibit wild-type virulence in C57BL/6 and BALB/c mice, but the B. abortus ahpC katE double mutant is extremely attenuated, and this attenuation is not relieved in derivatives of C57BL/6 mice that lack NADPH oxidase (cybb) or inducible nitric oxide synthase (Nos2) activity. These experimental findings indicate that the generation of endogenous H2O2 represents a relevant environmental stress that B. abortus 2308 must deal with during its residence in the host and that AhpC and KatE perform compensatory roles in detoxifying this metabolic H2O2.

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase.

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.

Establishment of a method for evaluating intracellular antibiotic efficacy in Brucella abortus-infected Mono Mac 6 monocytes.

BACKGROUND: Brucellae produce chronic and often lifelong infections in natural hosts. The persistent nature of these infections is predominantly due to the capacity of these bacteria to maintain intracellular residence in host macrophages. Successful antimicrobial therapy requires eradication of brucellae from this intracellular niche. It is important to seek new and improved antimicrobials for brucellosis therapy as well as a method to efficiently evaluate their intracellular efficacy. OBJECTIVES: For that reason, we have developed a method to evaluate intracellular drug efficacy for new and improved antimicrobials that show initial in vitro activity against Brucella species during drug screening. METHODS: Mono Mac 6 monocytes (MM6) were used because they are the only human cell line that constitutively expresses the phenotypic and functional characteristics of mature monocytes. This cell line has not previously been used with Brucella, therefore parallel studies were performed with J774 murine macrophages. Both cell lines were infected with Brucella abortus 2308 and antibiotics used clinically for treatment of brucellosis were used to determine intracellular efficacy. RESULTS: Significant differences in bacterial burden were observed at or above the MIC in both cell lines. Drug concentrations that fell below the MIC were found to significantly reduce intracellular brucellae only in MM6. CONCLUSIONS: The MM6 intracellular efficacy model will provide a useful method to examine the effect of novel antimicrobials for the treatment of human brucellosis.

Genetic basis for sulfonamide resistance in Bacillus anthracis.

Natural resistance of field strains of Bacillus anthracis to drugs from the sulfonamide class of antimicrobials that act by inhibiting dihydropteroate synthase (DHPS) has been reported. Though the structure of B. anthracis DHPS has been determined, its connection to the apparent intrinsic sulfonamide resistance of the bacterium has not been established. The aim of this study was to determine if a connection exists between DHPS and the observed sulfonamide resistance of B. anthracis. Microdilution broth assays verified that B. anthracis Sterne is highly resistant to a variety of sulfonamides with minimum inhibitory concentrations (MICs) exceeding 1250 microg/ml. A putative gene encoding DHPS (folP) was amplified from B. anthracis Sterne chromosomal DNA by polymerase chain reaction (PCR) and cloned. Sequence comparisons showed 100% identity with DHPSs from published genome sequences for various strains of B. anthracis. Additionally, expression of folP in B. anthracis Sterne was confirmed. Functionality of the B. anthracis DHPS was confirmed by complementation of an Escherichia coli folP deletion mutant as well as a standard enzyme assay. Concomitant transfer of high level sulfonamide resistance to this mutant along with increased sulfonamide IC(50)values for purified B. anthracis DHPS links DHPS to sulfonamide resistance in B. anthracis. These findings lay the groundwork that will aid future development of antimicrobics that target DHPS to treat anthrax infections.

Role of HdeA in acid resistance and virulence in Brucella abortus 2308.

Two-dimensional gel electrophoretic analysis of cell lysates suggests that stationary phase production of wild-type levels of an ortholog of the low pH dependent chaperone HdeA in Brucella abortus 2308 during growth in a minimal medium requires the presence of the RNA binding protein Hfq. Although mutational analysis demonstrated that HdeA contributes to acid resistance in this bacterium, this protein is not required for wild-type virulence in the BALB/c mouse model. These experimental findings indicate that the brucellae rely upon additional gene products to resist the acidic conditions they encounter in the phagosomal compartment of host macrophages.

The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice.

Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O(2)(-) generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O(2)(-) of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-gamma). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.

Intact purine biosynthesis pathways are required for wild-type virulence of Brucella abortus 2308 in the BALB/c mouse model.

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.

Adaptation of the Brucellae to their intracellular niche.

Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts.

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci.

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.