Biomedical subjects

Carlos J Orihuela

Publications and source records attributed to Carlos J Orihuela.

9 recordsLinked to original sources

PubMed2026

Biofilm formation during pneumococcal carriage imprints naturally acquired humoral immunity.

Streptococcus pneumoniae (Spn) colonization of the nasopharynx is a prerequisite for transmission and invasive disease. To investigate how repeated asymptomatic colonization shapes immunity and influences bacterial traits, we developed the Repeated Asymptomatic Murine Pneumococcal Colonization (RAMPC3) model using strains belonging to serotypes: 2 (D39), 3 (WU2), and 4 (TIGR4). Sequential colonization revealed strain- and exposure-order-dependent effects on bacterial burden, with initial colonization yielding robust carriage and subsequent exposures resulting in diminished burden and rapid clearance. Humoral profiling demonstrated antigenic imprinting: the first colonizing strain largely determined IgG and IgA specificity against bacterial proteins, with minimal diversification or expansion after repeated exposures. Reactivity was strongest for biofilm-associated antigens correlating with each strain's biofilm-forming capacity. Notably, experiments using human sera from naturally colonized adults mirrored these findings, with reactivity favoring biofilm antigens independent from capsule. Partial protection as result of colonization was demonstrated as triple-colonized mice had reduced mortality following pneumococcal pneumonia challenge. Likewise, mice colonized with biofilm deficient versions of TIGR4 and then challenged intratracheally with a serotype 6A (6A-10) strain were more likely to develop bacteremia, underscoring the contribution of the biofilm-associated host response to immunity. Finally, IgA responses in nasal-associated lymphoid tissue paralleled serum IgA patterns, validating systemic measurements as a proxy for mucosal immunity. These results reveal that biofilm formation during colonization is a key determinant of humoral immunity and contributes to systemic protection, providing insight into pneumococcal biology and informing strategies to design next-generation interventions.

Animals↗

PubMed2006

Platelet-activating factor receptor and innate immunity: uptake of gram-positive bacterial cell wall into host cells and cell-specific pathophysiology.

The current model of innate immune recognition of Gram-positive bacteria suggests that the bacterial cell wall interacts with host recognition proteins such as TLRs and Nod proteins. We describe an additional recognition system mediated by the platelet-activating factor receptor (PAFr) and directed to the pathogen-associated molecular pattern phosphorylcholine that results in the uptake of bacterial components into host cells. Intravascular choline-containing cell walls bound to endothelial cells and caused rapid lethality in wild-type, Tlr2(-/-), and Nod2(-/-) mice but not in Pafr(-/-) mice. The cell wall exited the vasculature into the heart and brain, accumulating within endothelial cells, cardiomyocytes, and neurons in a PAFr-dependent way. Physiological consequences of the cell wall/PAFr interaction were cell specific, being noninflammatory in endothelial cells and neurons but causing a rapid loss of cardiomyocyte contractility that contributed to death. Thus, PAFr shepherds phosphorylcholine-containing bacterial components such as the cell wall into host cells from where the response ranges from quiescence to severe pathophysiology.

Animals↗

PubMed2006

Cell wall-mediated neuronal damage in early sepsis.

Neuronal dysfunction can occur in the course of sepsis without meningitis. Sepsis-associated neuronal damage (SAND) was observed in the hippocampus within hours in experimental pneumococcal bacteremia. Intravascular challenge with purified bacterial cell wall recapitulated SAND. SAND persisted in PAFr(-/-) mice but was partially mitigated in mice lacking cell wall recognition proteins TLR2 and Nod2 and in mice overexpressing interleukin-10 (IL-10) in macrophages. Thus, cell wall drives SAND through IL-10-repressible inflammatory events. Treatment with CDP-choline ameliorated SAND, suggesting that it may be an effective adjunctive therapy to increase survival and reduce organ damage in sepsis.

Animals↗

PubMed2006

Identification of a Candidate Streptococcus pneumoniae core genome and regions of diversity correlated with invasive pneumococcal disease.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and gram-positive sepsis. While multiple virulence determinants have been identified, the combination of features that determines the propensity of an isolate to cause invasive pneumococcal disease (IPD) remains unknown. In this study, we determined the genetic composition of 42 invasive and 30 noninvasive clinical isolates of serotypes 6A, 6B, and 14 by comparative genomic hybridization. Comparison of the present/absent gene matrix (i.e., comparative genomic analysis [CGA]) identified a candidate core genome consisting of 1,553 genes (73% of the TIGR4 genome), 154 genes whose presence correlated with the ability to cause IPD, and 176 genes whose presence correlated with the noninvasive phenotype. Genes identified by CGA were cross-referenced with the published signature-tagged mutagenesis studies, which served to identify core and IPD-correlated genes required for in vivo passage. Among these, two pathogenicity islands, region of diversity 8a (RD8a), which encodes a neuraminidase and V-type sodium synthase, and RD10, which encodes PsrP, a protein homologous to the platelet adhesin GspB in Streptococcus gordonii, were identified. Mice infected with a PsrP mutant were delayed in the development of bacteremia and demonstrated reduced mortality versus wild-type-infected controls. Finally, the presence of seven RDs was determined to correlate with the noninvasive phenotype, a finding that suggests some RDs may contribute to asymptomatic colonization. In conclusion, RDs are unequally distributed between invasive and noninvasive isolates, RD8a and RD10 are correlated with the propensity of an isolate to cause IPD, and PsrP is required for full virulence in mice.

Adhesins, Bacterial↗

PubMed2005

Virulence in mice of pneumococcal clonal types with known invasive disease potential in humans.

Streptococcus pneumoniae isolates of serotypes 1, 4, 6B, 7F, 14, and 19F belonging to clonal types with known invasive disease potential in humans were used to infect C57BL/6 and BALB/c mice. Most isolates were able to colonize the nasopharynx for 7 days. One serotype 19F isolate of the clonal type ST162 had higher bacterial numbers than other isolates and clonal types of the same serotype. Serotype 4 clones caused the most-severe invasive disease, whereas serotype 1 clones caused low-level bacteremia without disease symptoms. BALB/c mice were more likely than C57BL/6 mice to develop meningitis. Disease kinetics varied significantly between clonal types. Although most induced a robust tumor necrosis factor response, some isolates of serotype 1 and 7F did not, suggesting that invasive disease caused by different clonal types may result in different degrees of host response. Capsular serotype, other clonal properties, and host factors are important for the development of pneumococcal disease.

Animals↗

PubMed2005

beta-Arrestin 1 participates in platelet-activating factor receptor-mediated endocytosis of Streptococcus pneumoniae.

Pneumococci traverse eukaryotic cells within vacuoles without intracytoplasmic multiplication. The platelet-activating factor receptor (PAFr) has been suggested as a portal of entry. Pneumococci colocalized with PAFr on endothelial cells and PAFr-/- mice showed a substantially impaired ability to support bacterial translocation, particularly from blood to brain. Pneumococci-induced colocalization of PAFr and beta-arrestin 1 at the plasma membrane of endothelial cells and PAFr-mediated pneumococcal uptake in transfected COS cells were greatly increased by cotransfection with the scaffold/adapter protein beta-arrestin 1. Activation of extracellular signal-regulated kinase kinases was required for uptake and was limited to the cytoplasmic compartment, consistent with activation by beta-arrestin rather than PAFr. Uptake of the pneumococcal vacuole involved clathrin, and half the bacteria proceeded into vacuoles marked by Rab5 and later Rab7, the classical route to the lysosome. Overexpression of beta-arrestin in endothelial cells decreased colocalization with Rab7. We conclude that the association of beta-arrestin with the PAFr contributes to successful translocation of pneumococci.

Animals↗

PubMed2004

Tissue-specific contributions of pneumococcal virulence factors to pathogenesis.

We assessed the ability of Streptococcus pneumoniae mutants deficient in either choline binding protein A (CbpA), pneumolysin (Pln), pyruvate oxidase (SpxB), autolysin (LytA), pneumococcal surface protein A, or neuraminidase A (NanA) to replicate in distinct anatomical sites and translocate from one site to the next. Intranasal, intratracheal, and intravenous models of disease were assessed in 4-week-old BALB/cJ mice by quantitation of bacterial titers in the relevant organs. Mice were also observed by use of real-time bioluminescent imaging (BLI). BLI allowed visualization of the bacteria in sites not tested by sampling. All mutants were created in D39 Xen7, a fully virulent derivative of capsular type 2 strain D39 that contains an optimized luxABCDE cassette. NanA, SpxB, and, to a lesser extent, CbpA contributed to prolonged nasopharyngeal colonization, whereas CbpA and NanA contributed to the transition to the lower respiratory tract. Once lung infection was established, Pln, SpxB, and LytA contributed to bacterial replication in the lungs and translocation to the bloodstream. In the bloodstream, only Pln and LytA were required for high-titer replication, whereas CbpA was required for invasion of the cerebrospinal fluid. We conclude that transitions between body sites require virulence determinants distinct from those involved in organ-specific replication.

Animals↗

PubMed2004

Microarray analysis of pneumococcal gene expression during invasive disease.

Streptococcus pneumoniae is a leading cause of invasive bacterial disease. This is the first study to examine the expression of S. pneumoniae genes in vivo by using whole-genome microarrays available from The Institute for Genomic Research. Total RNA was collected from pneumococci isolated from infected blood, infected cerebrospinal fluid, and bacteria attached to a pharyngeal epithelial cell line in vitro. Microarray analysis of pneumococcal genes expressed in these models identified body site-specific patterns of expression for virulence factors, transporters, transcription factors, translation-associated proteins, metabolism, and genes with unknown function. Contributions to virulence predicted for several unknown genes with enhanced expression in vivo were confirmed by insertion duplication mutagenesis and challenge of mice with the mutants. Finally, we cross-referenced our results with previous studies that used signature-tagged mutagenesis and differential fluorescence induction to identify genes that are potentially required by a broad range of pneumococcal strains for invasive disease.

Animals↗

PubMed2003

Organ-specific models of Streptococcus pneumoniae disease.

The variability of the course of infection by Streptococcus pneumoniae is well known but poorly understood. Most animal models of pneumonia, sepsis or meningitis have been forced to use site-specific bacterial inoculation to mimic localized human infection. This study examined the differences in the progression of disease-causing strains D39 (serotype 2), A66.1 (serotype 3) and TIGR4 (serotype 4) using isolates transformed with the Gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r). Expression of the lux operon results in bioluminescence, permitting the detection of the bacteria within a living animal while using a CCD camera. Mice infected intranasally with A66.1 developed only pneumonia, those challenged with D39 experienced high-grade sepsis, while TIGR4 infection resulted in low-grade pneumonia and bacteremia ultimately progressing to meningitis. Quantitative analysis of bacterial titers confirmed these patterns, which were consistent across different lineages of mice. Mice anesthetized with ketamine and xylazine developed more severe forms of the disease compared with isoflurane. These studies unambiguously characterize 3 distinct models of the natural course of pneumococcal infection. Mapping these models provides a framework for detailed molecular modeling of pneumococcal virulence determinants at specific stages of disease.

Animals↗