Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors. METHODS: We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE). RESULTS: In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination. CONCLUSION: These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections

The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus Ustilago maydis

In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast

Flagella and Motility in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate. In this work, it is demonstrated that A. pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins. The DNA sequence obtained through PCR of the fliC gene in A. pleuropneumoniae showed considerable identity (93%) in its 5′ and 3′ ends with the DNA sequences of corresponding genes in E. coli, Salmonella enterica, and Shigella spp. The motility of A. pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature. Flagella and motility may be involved in the survival and pathogenesis of A. pleuropneumoniae in pigs

Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics, participates in cell adhesion, and is present in biofilms

Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis

The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus <i>Ustilago maydis</i>

<div><p>In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus <i>Ustilago maydis</i>. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting <i>trt1</i> had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in <i>est2</i> budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of <i>trt1</i> crosses <i>in planta</i> suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.</p></div

Telomere repeat amplification protocol (TRAP) analysis in <i>U. maydis</i>.

<p>Telomerase activity in wild-type and mutant strains was determined. The absorbance data were used to construct a graphical representation of the telomerase activity for the sporidia of <i>U. maydis</i> strains (either wild-type or <i>trt</i>^-). Tumor cells derived from the 521×520 cross and a plant control were included to evaluate and detect telomerase activity. The medians of the telomerase-positive control cells (HEK293) and the 521 wild-type strain were significantly different from the median of the treated negative controls (P<0.05); however, no significant differences were detected between the negative controls and the <i>trt1</i>-disrupted mutants. The samples heated to 85°C are indicated with Δ, and the RNase-treated samples are designated as RNase. Telomerase activity was also determined in tumors and maize leaves.</p

<i>U. maydis</i> strains used.

<p>^*Partially characterized.</p><p><i>U. maydis</i> strains used.</p

Analysis of the effects of <i>trt</i>+ restoration in pTrt1 <i>U. maydis</i> transformants.

<p>The telomere length distribution in the telomerase-deficient strain W204 was assessed by Southern blotting TRF after reintroduction (200 doubling periods) of <i>tert1</i> in the pTrt1 <i>U. maydis</i> transformants as described above. (A) The TRF hybridization pattern of parental 521 (lane 1), 520 (lane 2), <i>trt1</i>-disrupted mutants trt1-1 (lane 3) and trt1-2 (lane 4) strains, the progeny derivative W204 (lane 5), and five of its W204-derived clones (T1 to T5, lanes 6 to 10) were analyzed using telomere sequences (TTAGGG) that were ³²P- labeled at 17 kBq/ml as probes. (C) The filter was stripped and re-hybridized to TR-p + TR-d sequences ³²P- labeled as a probe. The strain names are shown above the autoradiography. A molecular weight marker is shown on the left.</p