Increased Brucella abortus asRNA_0067 expression under intraphagocytic stressors is associated with enhanced virB2 transcription.

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system.

BACKGROUND: Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown. RESULTS: Re-examination of the ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium. CONCLUSIONS: Based on these results, we concluded that the operon ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.

Epitopes for Multivalent Vaccines Against Listeria, Mycobacterium and Streptococcus spp: A Novel Role for Glyceraldehyde-3-Phosphate Dehydrogenase.

The glycolytic enzyme and bacterial virulence factor of Listeria monocytogenes, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Lmo2459), ADP-ribosylated the small GTPase, Rab5a, and blocked phagosome maturation. This inhibitory activity localized within the NAD binding domain of GAPDH at the N-terminal 1-22 peptides, also conferred listeriosis protection when used in dendritic cell-based vaccines. In this study, we explore GAPDH of Listeria, Mycobacterium, and Streptococcus spp. taxonomic groups to search for epitopes that confer broad protection against pathogenic strains of these bacteria. GAPDH multivalent epitopes are selected if they induce inhibitory actions and wide-ranging immune responses. Proteomic isolation of GAPDH from dendritic cells infected with Listeria, Mycobacterium, or Streptococcus confirmed similar enzymatic, Rab5a inhibitory and immune stimulation abilities. We identified by bioinformatics and functional analyses GAPDH N-terminal 1-22 peptides from Listeria, Mycobacterium, and Streptococcus that shared 95% sequence homology, enzymatic activity, and B and T cell immune domains. Sera obtained from patients or mice infected with hypervirulent pathogenic Listeria, Mycobacterium, or Streptococcus presented high levels of anti-GAPDH 1-22 antibodies and Th2 cytokines. Monocyte derived dendritic cells from healthy donors loaded with GAPDH 1-22 peptides from Listeria, Mycobacterium, or Streptococcus showed activation patterns that correspond to cross-immunity abilities. In summary, GAPDH 1-22 peptides appeared as putative candidates to include in multivalent dendritic based vaccine platforms for Listeria, Mycobacterium, or Streptococcus.

Polymorphisms in Brucella Carbonic Anhydrase II Mediate CO2 Dependence and Fitness in vivo.

Some Brucella isolates are known to require an increased concentration of CO2 for growth, especially in the case of primary cultures obtained directly from infected animals. Moreover, the different Brucella species and biovars show a characteristic pattern of CO2 requirement, and this trait has been included among the routine typing tests used for species and biovar differentiation. By comparing the differences in gene content among different CO2-dependent and CO2-independent Brucella strains, we have confirmed that carbonic anhydrase (CA) II is the enzyme responsible for this phenotype in all the Brucella strains tested. Brucella species contain two CAs of the ß family, CA I and CA II; genetic polymorphisms exist for both of them in different isolates, but only those putatively affecting the activity of CA II correlate with the CO2 requirement of the corresponding isolate. Analysis of these polymorphisms does not allow the determination of CA I functionality, while the polymorphisms in CA II consist of small deletions that cause a frameshift that changes the C-terminus of the protein, probably affecting its dimerization status, essential for the activity. CO2-independent mutants arise easily in vitro, although with a low frequency ranging from 10-6 to 10-10 depending on the strain. These mutants carry compensatory mutations that produce a full-length CA II. At the same time, no change was observed in the sequence coding for CA I. A competitive index assay designed to evaluate the fitness of a CO2-dependent strain compared to its corresponding CO2-independent strain revealed that while there is no significant difference when the bacteria are grown in culture plates, growth in vivo in a mouse model of infection provides a significant advantage to the CO2-dependent strain. This could explain why some Brucella isolates are CO2 dependent in primary isolation. The polymorphism described here also allows the in silico determination of the CO2 requirement status of any Brucella strain.

Evaluation of the effects of erythritol on gene expression in Brucella abortus.

Bacteria of the genus Brucella have the unusual capability to catabolize erythritol and this property has been associated with their virulence mainly because of the presence of erythritol in bovine foetal tissues and because the attenuated S19 vaccine strain is the only Brucella strain unable to oxydize erythritol. In this work we have analyzed the transcriptional changes produced in Brucella by erythritol by means of two high throughput approaches: RNA hybridization against a microarray containing most of Brucella ORF's constructed from the Brucella ORFeome and next generation sequencing of Brucella mRNA in an Illumina GAIIx platform. The results obtained showed the overexpression of a group of genes, many of them in a single cluster around the ery operon, able to co-ordinately mediate the transport and degradation of erythritol into three carbon atoms intermediates that will be then converted into fructose-6P (F6P) by gluconeogenesis. Other induced genes participating in the nonoxidative branch of the pentose phosphate shunt and the TCA may collaborate with the ery genes to conform an efficient degradation of sugars by this route. On the other hand, several routes of amino acid and nucleotide biosynthesis are up-regulated whilst amino acid transport and catabolism genes are down-regulated. These results corroborate previous descriptions indicating that in the presence of erythritol, this sugar was used preferentially over other compounds and provides a neat explanation of the the reported stimulation of growth induced by erythritol.

Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium.

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

Characterization of defective beta-lactamase genes in Yersinia enterocolitica.

OBJECTIVES: To study at the molecular level the heterogeneity of expression of the two chromosomal beta-lactamases, BlaA and BlaB, in Yersinia enterocolitica strains isolated from clinical samples. METHODS: MIC determination by the agar dilution method and beta-lactamase assays was performed to determine the resistance level conferred by these enzymes. DNA cloning, PCR and direct sequencing were used to detect the presence of mutations. RESULTS: The blaA allele from strain IP97 (blaA97) was found to carry a deletion of 51 bp which entirely abolished its beta-lactamase activity. Both the ampR gene and the promoter region of strain Y56 were shown to be functional by a gene swapping experiment. The blaB allele from strain Y56 was found to carry two point mutations, only one of them resulting in a change in the amino acid sequence of the protein. This single amino acid change created a practically inactive BlaB or AmpC cephalosporinase in Y. enterocolitica Y56. CONCLUSIONS: The lack of activity observed in the beta-lactamases of some Y. enterocolitica isolates was due to the presence of point mutations or small deletions in the corresponding genes.

Tandem amplification of a 28-kilobase region from the Yersinia enterocolitica chromosome containing the blaA gene.

Most Yersinia enterocolitica strains are resistant to beta-lactam antibiotics due to the production of one or two chromosomally encoded beta-lactamases. Strain Y56 is a Y. enterocolitica O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A beta-lactamase. To select mutants with increased levels of resistance to beta-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 micro g of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a blaA-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the blaA gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the blaA locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event.

Targets for pSAM2 integrase-mediated site-specific integration in the Mycobacterium smegmatis chromosome.

An improved integrative cassette from plasmid pSAM2 has been constructed containing plasmid int and attP genes but excluding the xis gene, which should results in increased stability by suppression of the excision reaction. This cassette was included in both suicide and thermosensitive plasmids and used for integration in Mycobacterium smegmatis. Suicide plasmids containing this cassette integrated at a single site (attB1) in the M. smegmatis chromosome. The sequence of the attB1 site has been determined and was identified as a putative tRNA(Pro) gene. Thermosensitive plasmids containing the cassette integrated both at the same attB1 site and at other different sites, often giving rise to simultaneous integration at two sites. A second integration site (attB2) has been sequenced, which was located in the region encoding 16S rRNA of one of the two rrn operons of M. smegmatis.

The aquaporin gene aqpX of Brucella abortus is induced in hyperosmotic conditions.

An aquaporin gene (aqpX) was previously detected in the pathogenic bacterium Brucella abortus. Earlier studies showed that AqpX mediated rapid and large water fluxes in both directions in response to sudden osmotic up- or downshifts. Here, to study the role and the expression of the aqpX gene in B. abortus, an aqpX null mutant was constructed using an aqpX : : lacZ gene fusion. This mutant showed no significant difference in growth rate compared to the wild-type strain when grown in rich and minimal media, demonstrating that disruption of the aqpX gene was not lethal for B. abortus. The role of the B. abortus AqpX water channel was investigated by exposing the cells to hypo- and hyperosmolar conditions. While in hyperosmolar environments the growth rate of the knockout mutant was not affected, in hypo-osmolar conditions this mutant showed reduced viability after 50 h of growth. beta-Galactosidase assays and RT-PCR revealed that aqpX gene expression and the amount of aqpX mRNA were markedly increased in hyperosmolar conditions. Moreover, B. abortus aqpX expression levels were enhanced during the mid-exponential phase of growth. These results indicated that the expression of aqpX was regulated during the growth curve and induced in hyperosmolar conditions. This report is believed to be the first example of the induction of a bacterial aquaporin in hypertonic conditions.