Brucella lipopolysaccharide acts as a virulence factor

Brucella is a facultative intracellular bacterium responsible for brucellosis. Virulence factors involved in Brucella replication and Brucella's strategies to circumvent the immune response are under investigation. VirB proteins that form the type IV secretion system and that are involved in intracellular replication are considered as one of Brucella's virulence factors. In addition to this secretion system, bacterial outer membrane components have also been described as being implicated in Brucella survival in the host. For example, this bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. These properties make lipopolysaccharide an important virulence factor for Brucella survival and replication in the host.     

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.     

Age as a major factor associated with zinc and copper deficiencies in pediatric thalassemia

BackgroundPatients with thalassemia encounter increased consumption of zinc (Zn) and copper (Cu) from chronic hemolysis and increased excretion from iron chelation. Iron-enriched diet restriction may result in low Zn and Cu intakes. Recent data on Zn and Cu status among Thai pediatric patients with thalassemia are lacking. This study aimed to identify frequencies and determine risk factors of Zn and Cu deficiencies among patients with thalassemia.MethodsPatients with transfusion-dependent thalassemia (TDT) receiving iron chelation ≥12 months and nonTDT (NTDT) aged 2–20 years were recruited. Serum Zn and Cu were measured. Dietary intakes were ascertained by interviews.ResultsA total of 209 patients (TDT = 126, NTDT = 83) were enrolled. Zn deficiency seemed to be associated with disease severity as median (IQR) Zn level of TDT was lower than that of NTDT [77 (69−85) vs. 80 (72−88) mcg/dL, p = 0.05], while higher frequency of Zn deficiency was identified in the former (24 % vs. 14 %). In TDT, Zn deficiency was associated with patients >10 years (OR 4.6; 95 %CI 1.1–6.4, p = 0.03), which likely resulted from combined low dietary Zn intake, prolonged exposures to hemolysis and iron chelators. Frequencies of Cu deficiency were similarly low in TDT and NTDT (8% and 7%) with comparable median (IQR) Cu levels of 103 (90−124) and 110 (92−132) mcg/dL, respectively (p = 0.13). Cu levels were inversely associated with age (r=-0.65 and r=-0.62 in TDT and NTDT, respectively; p < 0.001).ConclusionCompared with younger patients, Zn and Cu deficiencies were more common among patients with thalassemia >10 years. Age was a major factor associated with both Zn and Cu deficiencies.     

Chronic injury as a major factor in susceptibility to gastric cancer

The etiology of gastric cancer is still unclear. The lesser curvature of stomach is more susceptible to chronic injury due to the anatomical characteristics. Many previous studies demonstrated that the lesser curvature is the most frequent tumor site in gastric cancer. And, precancerous gastric lesions, such as atrophic and intestinal metaplasia were also typically located in the lesser curvature. The lesser curvature is the first part of the stomach to be infected with Helicobacter pylori (H. pylori) infection which was associated with precancerous gastric lesions and gastric cancer. So, chronic injury of the stomach may lead to gastric cancer have hypothesized.     

Identification of ABCG2 dysfunction as a major factor contributing to gout

The ATP-binding cassette, subfamily G, member 2 gene ABCG2/BCRP locates in a gout-susceptibility locus (MIM 138900) on chromosome 4q. Recent genome-wide association studies also showed that the ABCG2 gene relates to serum uric acid levels and gout. Since ABCG2 is also known as a transporter of nucleotide analogs that are structurally similar to urate, and is an exporter that has common polymorphic reduced functionality variants, ABCG2 could be a urate secretion transporter and a gene causing gout. To find candidate mutations in ABCG2, we performed a mutation analysis of the ABCG2 gene in 90 Japanese patients with hyperuricemia and found six non-synonymous mutations. Among the variants, ATP-dependent urate transport was reduced or eliminated in five variants, and two out of the five variants (Q126X and Q141K) were frequently detected in patients. Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. As Q126X and Q141K are a nonfunctional and half-functional haplotype, respectively, their genotype combinations are divided into four estimated functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those who had dysfunctional ABCG2 had an increased risk of gout, and that a remarkable risk was observed in those with ≤1/4 function (OR, 25.8; 95% CI, 10.3-64.6; p = 3.39 × 10(-21)). In 2,150 Japanese individuals, the frequency of those with dysfunctional ABCG2 was more than 50%. Our function-based clinicogenetic analysis identified the combinations of dysfunctional variants of ABCG2 as a major contributing factor in Japanese patients with gout.     

Isolation and characterization of functional Leishmania major virulence factor UDP-galactopyranose mutase

Human parasitic pathogens of the genus Leishmania are the causative agents of cutaneous, mucocutaneous, and visceral leishmaniasis. Currently, there are millions of people infected with these diseases and over 50,000 deaths occur annually. Recently, it was shown that the flavin-dependent enzyme UDP-galactopyranose mutase (UGM) is a virulence factor in Leishmania major. UGM catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose. The product, UDP-galactofuranose, is the only source of galactofuranose which is present on the cell surface of this parasite and has been implicated to be important for host-parasite interactions. The recombinant form of this enzyme was obtained in a soluble and active form. The enzyme was shown to be active only in the reduced state. A k_cat value of 5 ± 0.2 s⁻¹ and a K_M value of 87 ± 11 μM were determined with UDP-galactofuranose as the substrate. Different from the dimeric bacterial and tetrameric fungal UGMs, this parasitic enzyme functions as a monomer.     

Phospholipases as possible virulence factor in Vibrio parahaemolyticus

Phospholipase C activity was elevated in pathogenic Vibrio parahaemolyticus isolated from patients. Phospholipase A activity was more pronounced in the nonpathogenic V. parahaemolyticus strains isolated from water. Extracts of the strains containing phospholipase C and A activity but no thermostable direct haemolysin (TDH) were capable of producing lesions in guinea pig skin indicating the presence of a toxic factor other than TDH. It is suggested that the toxic factor may be phospholipase C since the purified enzyme from Clostridium perfringens produced a similar reaction in guinea pig skin.     

Control of virulence by the two-component system CiaR/H is mediated via HtrA, a major virulence factor of Streptococcus pneumoniae

The CiaR/H two-component system is involved in regulating virulence and competence in Streptococcus pneumoniae. The system is known to regulate many genes, including that for high-temperature requirement A (HtrA). This gene has been implicated in the ability of the pneumococcus to colonize the nasopharynx of infant rats. We reported previously that deletion of the gene for HtrA made the pneumococcal strains much less virulent in mouse models, less able to grow at higher temperatures, and more sensitive to oxidative stress. In this report, we show that the growth phenotype as well as sensitivity to oxidative stress of Delta ciaR mutant was very similar to that of a Delta htrA mutant and that the expression of the HtrA protein was reduced in a ciaR-null mutant. Both the in vitro phenotype and the reduced virulence of Delta ciaR mutant could be restored by increasing the expression of HtrA.     

Staphylococcal DNA as a basis for classification

The genome characterization of the typing strains for all 13 species of the genus Staphylococcus, included into the Approval List of the Names of Bacterial (1980), is presented. The nucleotide composition of DNA (28-33% of GC) did not permit the differentiation between staphylococcal species, but some of the groups of these species could be differentiated by the size of their genome (0.6-1.6 X 10(9) daltons). Differences in the degree of similarity between the nucleotide sequences of the DNA of all species (5-6% of DNA homology) made it possible to suggest raising the genus Staphylococcus to the rank of the family Staphylococcaceae fam. nov. The hypothetical classification scheme of this family is presented for discussion.     

Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.     

Mycolic acid-containing glycolipid as a possible virulence factor of Rhodococcus equi for mice

By the use of various Rhodococcus equi strains differing in the length of carbon chains of glycolipid, we examined whether the glycolipid, glucose monomycolate, was contributing to the virulence of R. equi for mice. R. equi strains with longer carbon chain mycolic acid showed a higher virulence as determined by lethality and granuloma formation in mice than those with shorter ones. When purified glycolipid was injected into mice, granuloma formation and liver damage were most prominent with the glycolipid having longer carbon chain mycolic acid. Only a representative strain with longer carbon chain mycolic acid persisted in the spleen of mice after intravenous injection, while a strain with shorter carbon chain mycolic acid was readily eliminated. These results suggested that glycolipid was at least one of the virulence factors of R. equi and that the carbon chain length of mycolic acid might be critical in the expression of virulence.     

Application of immuno-mass spectrometry to analysis of a bacterial virulence factor

Here we describe a novel antibody-based assay that combines specificity of antibody with precision of mass spectral analysis. The assay is carried out in three steps using a single antigen capture and transfer reagent. The first step of the assay involves antibody immobilization. The second step is antigen capture and washing to remove unbound proteins. The third step involves the analysis of the captured antigens by surface enhanced laser desorption ionization time-of-flight mass spectrometry. The assay is facilitated by the ability of a single nonviable bacterial preparation expressing immunoglobulin-binding proteins that enable antibody immobilization, specific capture of fluid-phase antigen, and direct sample transfer to a protein chip for mass spectral analysis. Proof-of-concept studies using a model Streptococcus pyogenes virulence factor, the secreted cysteine protease SpeB, are presented.     

Role of the 25 kDa major outer membrane protein of Legionella pneumophila in attachment to U-937 cells and its potential as a virulence factor for chick embryos

The gene encoding the 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila was transformed into Escherichia coli JM 83 and the resultant E. coli LP 116 clone expressed the Legionella-MOMP. Compared with the parent E. coli strain, the clone showed a fivefold increase in opsonin-independent binding to U-937 cells. Furthermore, this gene was incorporated by electroporation into a low virulence derivative of Leg. pneumophila which showed reduced expression of the MOMP but enhanced expression of a 31 kDa protein in the OMP profile. After electroporation, the attenuated strain showed an increased expression of the MOMP while the 31 kDa protein was eliminated and virulence for the chick embryo was re-established. The use of a monoclonal antibody specific for the MOMP abolished virulence and adherence. These studies suggest that the 25 kDa MOMP of Leg. pneumophila serves as an adhesive molecule for host cells and that this protein plays a major role in the virulence of the organism for the chick embryo.     

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.