R. Martin Roop

Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host.

Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlies their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts, and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.

Interactions between Brucella melitensis and Human Phagocytes: Bacterial Surface O-Polysaccharide Inhibits Phagocytosis, Bacterial Killing, and Subsequent Host Cell Apoptosis

ABSTRACT Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth, virulent B. melitensis 16M results in a rough mutant designated WRR51. Unlike B. abortus, both smooth and rough strains of B. melitensis are resistant to complement-mediated killing. To determine the role of surface OPS in the interactions of B. melitensis with monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. Smooth B. melitensis survived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on live B. melitensis benefits the bacterium by preventing the death of macrophages, Brucellas preferred target for intracellular replication.

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

ABSTRACT 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 O2− generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O2− 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-γ). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-γ-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.

The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages.

The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.

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.

Subversion of Innate Immune Responses by Brucella through the Targeted Degradation of the TLR Signaling Adapter, MAL

Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis, and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian Toll/IL-1 receptor domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MyD88-adapter–like (MAL). This effect of TcpB is mediated through its box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TIR-domain containing adapter protein-inducing IFNβ. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly, the presence of TcpB leads to enhanced polyubiqitination of MAL, which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore, TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.

The Manganese Transporter MntH Is a Critical Virulence Determinant for Brucella abortus 2308 in Experimentally Infected Mice

ABSTRACT The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe2+, Zn2+, Co2+, or Ni2+. Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B. abortus 2308 is repressed by Mn2+, but not Fe2+, and this Mn-responsive expression is mediated by a Mur-like repressor. The B. abortus mntH mutant MWV15 exhibits increased susceptibility to oxidative killing in vitro compared to strain 2308, and a comparative analysis of the superoxide dismutase activities present in these two strains indicates that the parental strain requires MntH in order to make wild-type levels of its manganese superoxide dismutase SodA. The B. abortus mntH mutant also exhibits extreme attenuation in both cultured murine macrophages and experimentally infected C57BL/6 mice. These experimental findings indicate that Mn2+ transport mediated by MntH plays an important role in the physiology of B. abortus 2308, particularly during its intracellular survival and replication in the host.

Identification of two small regulatory RNAs linked to virulence in Brucella abortus 2308

Hfq is an RNA‐binding protein that functions in post‐transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over‐produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two‐plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.

Intact Purine Biosynthesis Pathways Are Required for Wild-Type Virulence of Brucella abortus 2308 in the BALB/c Mouse Model

ABSTRACT 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.

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.