-
[show abstract]
[hide abstract]
ABSTRACT: Background. Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo. Methods. We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models. Results. Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain. Conclusions. Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.
The Journal of Infectious Diseases 08/2012; 206(8):1185-93. · 6.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are frequently associated with strains harboring genes encoding Panton-Valentine leukocidin (PVL). The role of PVL in the success of the epidemic CA-MRSA strain USA300 remains unknown. Here we developed a skin and soft tissue infection model in rabbits to test the hypothesis that PVL contributes to USA300 pathogenesis and compare it with well-established virulence determinants: alpha-hemolysin (Hla), phenol-soluble modulin-alpha peptides (PSMα), and accessory gene regulator (Agr). The data indicate that Hla, PSMα, and Agr contribute to the pathogenesis of USA300 skin infections in rabbits, whereas a role for PVL could not be detected.
The Journal of Infectious Diseases 09/2011; 204(6):937-41. · 6.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. We tested the hypothesis that alpha-hemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Deltahla) strains caused skin lesions in a mouse infection model that were significantly smaller than those caused by wild-type USA300 and Newman strains. Moreover, infection due to wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Deltahla strains. Passive immunization with Hla-specific antisera or active immunization with a nontoxigenic form of Hla significantly reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude that Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections.
The Journal of Infectious Diseases 10/2010; 202(7):1050-8. · 6.41 Impact Factor
-
Gail L Sturdevant,
Laszlo Kari, Donald J Gardner,
Norma Olivares-Zavaleta,
Linnell B Randall,
William M Whitmire,
John H Carlson,
Morgan M Goheen,
Elizabeth M Selleck,
Craig Martens,
Harlan D Caldwell
[show abstract]
[hide abstract]
ABSTRACT: Chlamydia trachomatis is a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model of C. trachomatis infection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivated in vitro prior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiple in vitro passages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrasting in vivo virulence characteristics, the strains exhibited no differences in the results of in vitro infectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.
Infection and immunity 09/2010; 78(9):3660-8. · 4.21 Impact Factor
-
Binh An Diep,
Liana Chan,
Pierre Tattevin,
Osamu Kajikawa,
Thomas R. Martin,
Li Basuino,
Thuy T. Mai,
Helene Marbach,
Kevin R. Braughton,
Adeline R. Whitney, Donald J. Gardner,
Xuemo Fan,
Ching W. Tseng,
George Y. Liu,
Cedric Badiou,
Jerome Etienne,
Gerard Lina,
Michael A. Matthay,
Frank R. DeLeo,
Henry F. Chambers
[show abstract]
[hide abstract]
ABSTRACT: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300,
like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear
leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection
models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like
those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect
results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of
a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of
USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features
of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by
recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights
into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated
pathogenesis.
Proceedings of the National Academy of Sciences 03/2010; 107(12):5587-5592. · 9.68 Impact Factor
-
Randall J Olsen,
Scott D Kobayashi,
Ara A Ayeras,
Madiha Ashraf,
Shawna F Graves,
Willie Ragasa,
Tammy Humbird,
Jamieson L Greaver,
Constance Cantu,
Jody L Swain,
Leslie Jenkins,
Terry Blasdel,
Philip T Cagle, Donald J Gardner,
Frank R DeLeo,
James M Musser
[show abstract]
[hide abstract]
ABSTRACT: Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureus virulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureus strain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureus pneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.
American Journal Of Pathology 03/2010; 176(3):1346-54. · 4.89 Impact Factor
-
Patrick R Shea,
Kimmo Virtaneva,
John J Kupko,
Stephen F Porcella,
William T Barry,
Fred A Wright,
Scott D Kobayashi,
Aaron Carmody,
Robin M Ireland,
Daniel E Sturdevant,
Stacy M Ricklefs,
Imran Babar,
Claire A Johnson,
Morag R Graham, Donald J Gardner,
John R Bailey,
Michael J Parnell,
Frank R Deleo,
James M Musser
[show abstract]
[hide abstract]
ABSTRACT: Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.
Proceedings of the National Academy of Sciences 02/2010; 107(10):4693-8. · 9.68 Impact Factor
-
Binh An Diep,
Liana Chan,
Pierre Tattevin,
Osamu Kajikawa,
Thomas R. Martin,
Li Basuino,
Thuy T. Mai,
Helene Marbach,
Kevin R. Braughton,
Adeline R. Whitney, Donald J. Gardner,
Xuemo Fan,
Ching W. Tseng,
George Y. Liu,
Cedric Badiou,
Jerome Etienne,
Gerard Lina,
Michael A. Matthay,
Frank R. DeLeo,
Henry F. Chambers
[show abstract]
[hide abstract]
ABSTRACT: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300, like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.
Proceedings of the National Academy of Sciences 01/2010; 107(12):5587. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: sThe role of Panton-Valentine leukocidin (PVL) in Staphylococcus aureus pathogenesis is controversial. Here, we show that an unintended point mutation in the agr P2 promoter of S. aureus caused the phenotypes in gene regulation and murine pneumonia attributed to PVL by earlier investigators. In agreement with other studies that failed to detect similar effects of PVL using community-associated methicillin-resistant S. aureus strains, we found no significant effect of PVL on gene expression or pathogenesis after we repaired the mutation. These findings provide further evidence that PVL does not have a major impact on S. aureus pathogenesis. Moreover, our results demonstrate that a single nucleotide polymorphism in an intergenic region can dramatically affect bacterial physiology and virulence. Finally, our work emphasizes the need to frequently evaluate the integrity of the S. aureus agr locus.
The Journal of Infectious Diseases 10/2009; 200(5):724-34. · 6.41 Impact Factor
-
Jovanka M Voyich,
Michael Otto,
Barun Mathema,
Kevin R Braughton,
Adeline R Whitney,
Diane Welty,
R Daniel Long,
David W Dorward, Donald J Gardner,
Gérard Lina,
Barry N Kreiswirth,
Frank R DeLeo
[show abstract]
[hide abstract]
ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) remains a major problem in hospitals, and it is now spreading in the community. A single toxin, Panton-Valentine leukocidin (PVL), has been linked by epidemiological studies to community-associated MRSA (CA-MRSA) disease. However, the role that PVL plays in the pathogenesis of CA-MRSA has not been tested directly. To that end, we used mouse infection models to compare the virulence of PVL-positive with that of PVL-negative CA-MRSA representing the leading disease-causing strains. Unexpectedly, strains lacking PVL were as virulent in mouse sepsis and abscess models as those containing the leukotoxin. Isogenic PVL-negative (lukS/F-PV knockout) strains of USA300 and USA400 were as lethal as wild-type strains in a sepsis model, and they caused comparable skin disease. Moreover, lysis of human neutrophils and pathogen survival after phagocytosis were similar between wild-type and mutant strains. Although the toxin may be a highly linked epidemiological marker for CA-MRSA strains, we conclude that PVL is not the major virulence determinant of CA-MRSA.
The Journal of Infectious Diseases 01/2007; 194(12):1761-70. · 6.41 Impact Factor
-
Morag R Graham,
Kimmo Virtaneva,
Stephen F Porcella, Donald J Gardner,
R Daniel Long,
Diane M Welty,
William T Barry,
Claire A Johnson,
Larye D Parkins,
Fred A Wright,
James M Musser
[show abstract]
[hide abstract]
ABSTRACT: Molecular mechanisms mediating group A Streptococcus (GAS)-host interactions remain poorly understood but are crucial for diagnostic, therapeutic, and vaccine development. An optimized high-density microarray was used to analyze the transcriptome of GAS during experimental mouse soft tissue infection. The transcriptome of a wild-type serotype M1 GAS strain and an isogenic transcriptional regulator knockout mutant (covR) also were compared. Array datasets were verified by quantitative real-time reverse transcriptase-polymerase chain reaction and in situ immunohistochemistry. The results unambiguously demonstrate that coordinated expression of proven and putative GAS virulence factors is directed toward overwhelming innate host defenses leading to severe cellular damage. We also identified adaptive metabolic responses triggered by nutrient signals and hypoxic/acidic conditions in the host, likely facilitating pathogen persistence and proliferation in soft tissues. Key discoveries included that oxidative stress genes, virulence genes, genes related to amino acid and maltodextrin utilization, and several two-component transcriptional regulators were highly expressed in vivo. This study is the first global analysis of the GAS transcriptome during invasive infection. Coupled with parallel analysis of the covR mutant strain, novel insights have been made into the regulation of GAS virulence in vivo, resulting in new avenues for targeted therapeutic and vaccine research.
American Journal Of Pathology 10/2006; 169(3):927-42. · 4.89 Impact Factor
-
Jovanka M Voyich,
Kevin R Braughton,
Daniel E Sturdevant,
Adeline R Whitney,
Battouli Saïd-Salim,
Stephen F Porcella,
R Daniel Long,
David W Dorward, Donald J Gardner,
Barry N Kreiswirth,
James M Musser,
Frank R DeLeo
[show abstract]
[hide abstract]
ABSTRACT: Polymorphonuclear leukocytes (PMNs, or neutrophils) are critical for human innate immunity and kill most invading bacteria. However, pathogens such as Staphylococcus aureus avoid destruction by PMNs to survive, thereby causing human infections. The molecular mechanisms used by pathogens to circumvent killing by the immune system remain largely undefined. To that end, we studied S. aureus pathogenesis and bacteria-PMN interactions using strains originally isolated from individuals with community-acquired (CA) and hospital-acquired infections. Compared with strains from hospital infections (COL and MRSA252), strain MW2 and a methicillin-susceptible relative, MnCop, were significantly more virulent in a mouse model of S. aureus infection, and caused the greatest level of pathology in major vital organs. Although phagocytosis of each strain triggered production of reactive oxygen species and granule-phagosome fusion, those from CA infections were significantly more resistant to killing by human PMNs and caused greater host cell lysis. Microarray analysis of the strains during neutrophil phagocytosis identified genes comprising a global S. aureus response to human innate host defense. Genes involved in capsule synthesis, gene regulation, oxidative stress, and virulence, were up-regulated following ingestion of the pathogen. Notably, phagocytosis of strains from CA infections induced changes in gene expression not observed in the other strains, including up-regulation of genes encoding virulence factors and hypothetical proteins. Our studies reveal a gene transcription program in a prominent human pathogen that likely contributes to evasion of innate host defense.
The Journal of Immunology 10/2005; 175(6):3907-19. · 5.79 Impact Factor
-
Paul Sumby,
Kent D Barbian, Donald J Gardner,
Adeline R Whitney,
Diane M Welty,
R Daniel Long,
John R Bailey,
Michael J Parnell,
Nancy P Hoe,
Gerald G Adams,
Frank R Deleo,
James M Musser
[show abstract]
[hide abstract]
ABSTRACT: Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal- and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.
Proceedings of the National Academy of Sciences 03/2005; 102(5):1679-84. · 9.68 Impact Factor
-
Tamar Ben-Yosef,
Inna A Belyantseva,
Thomas L Saunders,
Elizabeth D Hughes,
Kohei Kawamoto,
Christina M Van Itallie,
Lisa A Beyer,
Kärin Halsey, Donald J Gardner,
Edward R Wilcox,
Julia Rasmussen,
James M Anderson,
David F Dolan,
Andrew Forge,
Yehoash Raphael,
Sally A Camper,
Thomas B Friedman
[show abstract]
[hide abstract]
ABSTRACT: Tight junctions (TJs) create ion-selective paracellular permeability barriers between extracellular compartments. In the organ of Corti of the inner ear, TJs of the reticular lamina separate K(+)-rich endolymph and Na(+)-rich perilymph. In humans, mutations of the gene encoding claudin 14 TJ protein cause profound deafness but the underlying pathogenesis is unknown. To explore the role of claudin 14 in the inner ear and in other tissues we created a mouse model by a targeted deletion of Cldn14. In the targeted allele a lacZ cassette is expressed under the Cldn14 promoter. In Cldn14-lacZ heterozygous mice beta-galactosidase activity was detected in cochlear inner and outer hair cells and supporting cells, in the collecting ducts of the kidney, and around the lobules of the liver. Cldn14-null mice have a normal endocochlear potential but are deaf due to rapid degeneration of cochlear outer hair cells, followed by slower degeneration of the inner hair cells, during the first 3 weeks of life. Monolayers of MDCK cells expressing claudin 14 show a 6-fold increase in the transepithelial electrical resistance by decreasing paracellular permeability for cations. In wild type mice, claudin 14 was immunolocalized at hair cell and supporting cell TJs. Our data suggest that the TJ complex at the apex of the reticular lamina requires claudin 14 as a cation-restrictive barrier to maintain the proper ionic composition of the fluid surrounding the basolateral surface of outer hair cells.
Human Molecular Genetics 09/2003; 12(16):2049-61. · 7.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Over a period of ten months, five mice submitted to our service (the Pathology Section of the Veterinary Resources Program, Office of Research Services at the National Institutes of Health, Bethesda, Md.) were diagnosed with disseminated trichosporonosis. These mice had pyogranulomatous inflammation in multiple organs, including lung, liver, lymph nodes, salivary gland, and skin. Fungal elements in many of the lesions were identified, using special histochemical stains, and Trichosporon beigelii was obtained by use of culture of specimens at affected sites. This saprophytic fungus has caused disseminated disease in immunosuppressed humans. However, despite widespread use of immunosuppressed rodents in research, to the authors' knowledge, this organism had not previously been reported to cause spontaneous disseminated disease in laboratory mice. All affected mice had a genetically engineered defect in p47(phox), a critical component of the nicotinamide dinucleotide phosphate (NADPH) oxidase, the enzyme responsible for generating the phagocyte oxidative burst. These animals are used as a murine model of human chronic granulomatous disease. We discuss the lesions, differential diagnosis, identification of the organism, and the role of NADPH oxidase in protecting against disseminated trichosporonosis.
Comparative medicine 07/2003; 53(3):303-8. · 1.05 Impact Factor
