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C J Orihuela

Publications and source records attributed to C J Orihuela.

4 recordsLinked to original sources
PubMed2001

Identification and characterization of an in vivo regulated D15/Oma87 homologue in Shigella flexneri using differential display polymerase chain reaction.

Shigella genes expressed during infection likely contribute to adaptation and virulence in the host. Using differential display PCR (DDPCR), a cDNA fragment from Shigella flexneri serotype 5 that showed enhanced expression in a murine model was identified, cloned and sequenced. Enhanced expression was verified by RNA dot blot. The full-length gene was cloned using PCR and sequenced. The complete gene sequence was BLAST searched against GenBank, and exhibited strong homology to genes encoding Haemophilus influenzae D15 and Pasteurella multocida Oma87 protective outer membrane antigens. The S. flexneri gene putatively encodes a approximately 90-kDa protein and was termed oma90. The deduced amino acid sequence from oma90 was analyzed and compared to the D15/Oma87 antigens. Additionally, oma90 mapped to a cluster of orthologous groups, and probably contains an ancient conserved domain. The chromosomal organization of oma90 was similar to that for H. influenzae and P. multocida as well as for other known homologues. Northern blot revealed that the oma90 transcript encoded only oma90. This report represents the first description of a S. flexneri gene identified based on enhanced expression in the host. Furthermore, we report the first evidence demonstrating in vivo regulation of a member of the d15/oma87 gene family.

Amino Acid Sequence↗
PubMed2001

Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity.

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

ATP-Binding Cassette Transporters↗
PubMed2000

Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties.

We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

Animals↗