Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

Phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan.

The purposes of this study were to determine phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan and to determine their biotype. The focus was laid on culture-morphological, biochemical, and biological properties of 59 Brucella isolates from primary cultures. Material was isolated from blood and tissue of serum-positive killed, dead diseased, or aborted domestic cattle from different regions of Kazakhstan where brucellosis is a common problem. Multiple-locus variable number tandem repeat analysis (MLVA) of all strains, isolated in different regions, has shown that Brucella isolates from the epizootic form two clusters. Based on the comparison with strains available in the MLVA database, B. abortus 0015/B is alike the B. abortus strains isolated from Italy and Portugal. B. melitensis 0016/B isolated from the Almaty region fits the third cluster and is alike the B. melitensis strains isolated from humans in Turkey, China, and Portugal. More than 90% of the overall B. abortus samples were isolated from the northern regions of the East and West Kazakhstan, while B. melitensis strains were registered in the southeast Kazakhstan. The most frequently recorded B. abortus biovar is biovar 3. The most frequently recorded B. melitensis biovars are biovars 1 and 3. SIGNIFICANCE AND IMPACT OF STUDY: These results contribute to a better understanding of the geographic pattern of Brucella infection in Kazakh cattle also important for developing the specific control measures. The results of current research can be used for creating a gene bank of Brucella strains circulating in Kazakhstan for producing diagnostic and therapeutic agents. The research material will be used to solve the problems of genetic characterization of Brucella species and to establish the phylogenetic relationships of strains.

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum.

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.

OtpR regulated the growth, cell morphology of B. melitensis and tolerance to ß-lactam agents.

The intracellular pathogen, Brucella melitensis, possesses an operon with two components: otpR (BMEI0066), which encodes a response regulator, and BMEI0067, which encodes a putative cAMP-dependent protein kinase regulatory subunit. Previous studies have shown that a polar mutation in the BMEI0066 gene significantly decreased virulence and stress tolerance in Brucella. In this study, we constructed non-polar mutant with deletion of otpR, as well as its complementary strain to further investigate the function of otpR. The ΔotpR mutant produced smaller colonies on TSA plates, and grew slower in tryptic soy broth compared to 16M or the otpR-complemented strain CotpR. Electron microscopy revealed that ΔotpR displayed an unusual, irregular deformation of the cell surface in contrast to the native coccobacillus shape of 16M. These results showed that OtpR played a key role in the maintenance of cell shape. To determine the effect of the otpR mutant on antibiotic susceptibility, compared the parent strain, the mutant was two- to eight-fold more susceptible to all the ß-lactam antibiotics tested. Furthermore, comparative real-time qPCR of genes that related to penicillin binding proteins of cell wall synthesis and cell division showed that the otpR mutation resulted in reduced expression of pbp1C, pbp6B, pbp6C and ftsQ. Taken together, these data revealed that the OtpR activity is necessary for growth, and cell morphology and tolerance to ß-lactam agents of B. melitensis.

The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection.

Persistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as non-motile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues.

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.

The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity.

Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 microg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to beta-lactam antibiotics. PBP2 complements the activity of PBP1. New beta-lactam antibiotics and improved vaccines might be developed based on this property.

Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes.

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

Immunogenic properties of Brucella melitensis cell-wall fractions in BALB/c mice.

The immunogenicity of several Brucella melitensis cell-wall (CW) fractions was tested in BALB/c mice. These CW fractions were smooth lipopolysaccharide (S-LPS) fraction from smooth (S) B. melitensis strain 16M, sodium dodecyl sulphate-insoluble (SDS-I) CW fraction from B. melitensis strain 16M (S) undigested or digested with pepsin, and SDS-I CW fraction from rough (R) B. melitensis strain H38. The B. melitensis SDS-I CW fraction contained two major outer-membrane proteins (OMPs) of 25-27 kDa and 31-34 kDa, peptidoglycan (PG) and a small quantity (1.5%) of LPS. One month after immunisation, mice were challenged with virulent B. melitensis strain H38 (S) and Brucella spleen counts were recorded on days 28 and 49 after challenge. Before challenge, as measured by ELISA, the highest antibody responses to S-LPS were observed in mice immunised with SDS-I CW fraction from B. melitensis strain 16M (S), whether digested with pepsin or undigested. All immunised mice, except those immunised with the SDS-I CW fraction from the R strain, showed higher IgG1 than IgG2a antibody responses to S-LPS (IgG1:IgG2a ratio 3.64-7.71). Antibody responses to the 25-27-kDa OMP were very low, with the highest responses in the mice immunised with the SDS-I CW fraction from the R strain. These results indicated that, in BALB/c mice, these CW fractions probably induced Th2-dependent more than Th1-dependent antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS)