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4. Generation of targeted Chlamydia trachomatis null mutants

Author

Kari, Laszlo, Goheen, Morgan M., Randall, Linnell B., Taylor, Lacey D., Carlson, John H., Whitmire, William M., Virok, Dezso, Rajaram, Krithika, Endresz, Valeria, McClarty, Grant, Nelson, David E., Caldwell, Harlan D., and Gotschlich, Emil C.

Published
2011

9. Chlamydial IFN-[gamma], immune evasion is linked to host infection tropism

Author

Nelson, David E., Virok, Dezso P., Wood, Heidi, Roshick, Christine, Johnson, Raymond M., Whitrnire, William M., Crane, Deborah D., Steele-Mortimer, Olivia, Kari, Laszlo, McClarty, Grant, and Caldwell, Harlan D.

Subjects
Chlamydia -- Research, Science and technology
Abstract

Chlamydiae are obligate intracellular pathogens that can exhibit a broad host range in infection tropism despite maintaining near genomic identity. Here, we have investigated the molecular basis for this unique host--pathogen relationship. We show that human and murine chlamydial infection tropism is linked to unique host and pathogen genes that have coevolved in response to host immunity. This intimate host--pathogen niche revolves around a restricted repertoire of host species-specific IFN-[gamma]-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-[gamma] induces indoleamine 2,3-dioxygenase expression that inhibits chlamydial growth by depleting host tryptophan pools. Human chlamydial strains, but not the mouse strain, avoid this response by the production of tryptophan synthase that rescues them from tryptophan starvation. Conversely, in murine epithelial cells IFN-[gamma] induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (ligp1) was shown by small interfering RNA silencing experiments to specifically inhibit human strains, but not the mouse strain. Like human strains and their host cells, the murine strain has coevolved with its murine host by producing a large toxin possessing YopT homology, possibly to circumvent host GTPases. Collectively, our findings show chlamydial host infection tropism is determined by IFN-[gamma]-mediated immunity. immunity | virulence factors | pathogenesis | defense | coevolution

Published
2005

11. Chlamydia trachomatis Plasmid Gene Protein 3 Is Essential for the Establishment of Persistent Infection and Associated Immunopathology

Author

Yang, Chunfu, primary, Kari, Laszlo, additional, Lei, Lei, additional, Carlson, John H., additional, Ma, Li, additional, Couch, Claire E., additional, Whitmire, William M., additional, Bock, Kevin, additional, Moore, Ian, additional, Bonner, Christine, additional, McClarty, Grant, additional, and Caldwell, Harlan D., additional

Published
2020
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16. Comprehensive global genome dynamics of show ancient diversification followed by contemporary mixing and recent lineage expansion

Author

Hadfield, James, Harris, Simon R, Seth-Smith, Helena M B, Parmar, Surendra, Andersson, Patiyan, Giffard, Philip M, Schachter, Julius, Moncada, Jeanne, Ellison, Louise, Vaulet, María Lucía Gallo, Fermepin, Marcelo Rodríguez, Radebe, Frans, Mendoza, Suyapa, Ouburg, Sander, Morré, Servaas A, Sachse, Konrad, Puolakkainen, Mirja, Korhonen, Suvi J, Sonnex, Chris, Wiggins, Rebecca, Jalal, Hamid, Brunelli, Tamara, Casprini, Patrizia, Pitt, Rachel, Ison, Cathy, Savicheva, Alevtina, Shipitsyna, Elena, Hadad, Ronza, Kari, Laszlo, Burton, Matthew J, Mabey, David, Solomon, Anthony W, Lewis, David, Marsh, Peter, Unemo, Magnus, Clarke, Ian N, Parkhill, Julian, Thomson, Nicholas R, and Medical Microbiology and Infection Prevention

Abstract

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis.

Published
2017
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19. Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion

Author

Hadfield, James, primary, Harris, Simon R., additional, Seth-Smith, Helena M.B., additional, Parmar, Surendra, additional, Andersson, Patiyan, additional, Giffard, Philip M., additional, Schachter, Julius, additional, Moncada, Jeanne, additional, Ellison, Louise, additional, Vaulet, María Lucía Gallo, additional, Fermepin, Marcelo Rodríguez, additional, Radebe, Frans, additional, Mendoza, Suyapa, additional, Ouburg, Sander, additional, Morré, Servaas A., additional, Sachse, Konrad, additional, Puolakkainen, Mirja, additional, Korhonen, Suvi J., additional, Sonnex, Chris, additional, Wiggins, Rebecca, additional, Jalal, Hamid, additional, Brunelli, Tamara, additional, Casprini, Patrizia, additional, Pitt, Rachel, additional, Ison, Cathy, additional, Savicheva, Alevtina, additional, Shipitsyna, Elena, additional, Hadad, Ronza, additional, Kari, Laszlo, additional, Burton, Matthew J., additional, Mabey, David, additional, Solomon, Anthony W., additional, Lewis, David, additional, Marsh, Peter, additional, Unemo, Magnus, additional, Clarke, Ian N., additional, Parkhill, Julian, additional, and Thomson, Nicholas R., additional

Published
2017
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20. Chlamydia trachomatis ChxR is a transcriptional regulator of virulence factors that function in in vivo host–pathogen interactions

Author

Yang, Chunfu, primary, Kari, Laszlo, additional, Sturdevant, Gail L., additional, Song, Lihua, additional, Patton, Michael John, additional, Couch, Claire E., additional, Ilgenfritz, Jillian M., additional, Southern, Timothy R., additional, Whitmire, William M., additional, Briones, Michael, additional, Bonner, Christine, additional, Grant, Chris, additional, Hu, Pinzhao, additional, McClarty, Grant, additional, and Caldwell, Harlan D., additional

Published
2017
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21. Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion

Author

Hadfield, James, Harris, Simon R, Seth-Smith, Helena M B, Parmar, Surendra, Andersson, Patiyan, Giffard, Philip M., Schachter, Julius, Moncada, Jeanne, Ellison, Louise, Vaulet, María Lucía Gallo, Fermepin, Marcelo Rodríguez, Radebe, Frans, Mendoza, Suyapa, Ouburg, Sander, Morré, Servaas A, Sachse, Konrad, Puolakkainen, Mirja, Korhonen, Suvi J, Sonnex, Chris, Wiggins, Rebecca, Jalal, Hamid, Brunelli, Tamara, Casprini, Patrizia, Pitt, Rachel, Ison, Cathy, Savicheva, Alevtina, Shipitsyna, Elena, Hadad, Ronza, Kari, Laszlo, Burton, Matthew J, Mabey, David, Solomon, Anthony W, Lewis, David, Marsh, Peter, Unemo, Magnus, Clarke, Ian N, Parkhill, Julian, Thomson, Nicholas R., Hadfield, James, Harris, Simon R, Seth-Smith, Helena M B, Parmar, Surendra, Andersson, Patiyan, Giffard, Philip M., Schachter, Julius, Moncada, Jeanne, Ellison, Louise, Vaulet, María Lucía Gallo, Fermepin, Marcelo Rodríguez, Radebe, Frans, Mendoza, Suyapa, Ouburg, Sander, Morré, Servaas A, Sachse, Konrad, Puolakkainen, Mirja, Korhonen, Suvi J, Sonnex, Chris, Wiggins, Rebecca, Jalal, Hamid, Brunelli, Tamara, Casprini, Patrizia, Pitt, Rachel, Ison, Cathy, Savicheva, Alevtina, Shipitsyna, Elena, Hadad, Ronza, Kari, Laszlo, Burton, Matthew J, Mabey, David, Solomon, Anthony W, Lewis, David, Marsh, Peter, Unemo, Magnus, Clarke, Ian N, Parkhill, Julian, and Thomson, Nicholas R.

Abstract

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis., Funding agencies:Sanger Institute through the Wellcome Trust 098051 Australian National Health and Medical Research Council 1060768

Published
2017
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24. Chlamydia trachomatis ChxR is a transcriptional regulator of virulence factors that function in in vivo host-pathogen interactions.

Author

Chunfu Yang, Kari, Laszlo, Sturdevant, Gail L., Lihua Song, Patton, Michael John, Couch, Claire E., Ilgenfritz, Jillian M., Southern, Timothy R., Whitmire, William M., Briones, Michael, Bonner, Christine, Grant, Chris, Pinzhao Hu, McClarty, Grant, and Caldwell, Harlan D.

Subjects
*CHLAMYDIA trachomatis, *CHLAMYDIA, *PATHOGENIC microorganisms, *MAMMALIAN cell cycle, *PROTEOMICS
Abstract

Chlamydia trachomatis is an obligate intracellular pathogen characterized by a unique biphasic developmental cycle that alternates between infectious and non-infectious organisms. Chlamydial ChxR is a transcriptional activator that has been implicated in the regulation of the development cycle. We used a reverse genetics approach to generate three chxR null mutants. All three mutants grew normally in cultured mammalian cells. Whole genome sequencing identified SNPs in other genes; however, none of the mutated genes were common to all three ChxR null mutants arguing against a genetic compensatory mechanism that would explain the non-essential in vitro growth phenotype. Comparative proteomics identified five proteins, CT005, CT214, CT565, CT694 and CT695, that were significantly downregulated in all ChxR null mutants. This group includes established inclusion membrane and type III secreted proteins. ChxR transcriptional regulation of these genes was confirmed by qRT-PCR. Importantly, while ChxR null mutants exhibited no growth deficiencies in in vitro, they did show significant differences in in vivo growth using a mouse genital tract model. Collectively, our findings demonstrated that ChxR is a transcriptional activator that regulates the expression of virulence genes whose functions are restricted to in vivo infection. [ABSTRACT FROM AUTHOR]

Published
2017
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27. Antibody Signature of Spontaneous Clearance of Chlamydia trachomatis Ocular Infection and Partial Resistance against Re-challenge in a Nonhuman Primate Trachoma Model

Author

Kari, Laszlo, primary, Bakios, Lauren E., additional, Goheen, Morgan M., additional, Bess, Leah N., additional, Watkins, Heather S., additional, Southern, Timothy R., additional, Song, Lihua, additional, Whitmire, William M., additional, Olivares-Zavaleta, Norma, additional, and Caldwell, Harlan D., additional

Published
2013
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29. Chlamydia trachomatis Plasmid-Encoded Pgp4 Is a Transcriptional Regulator of Virulence-Associated Genes

Author

Song, Lihua, primary, Carlson, John H., additional, Whitmire, William M., additional, Kari, Laszlo, additional, Virtaneva, Kimmo, additional, Sturdevant, Daniel E., additional, Watkins, Heather, additional, Zhou, Bing, additional, Sturdevant, Gail L., additional, Porcella, Stephen F., additional, McClarty, Grant, additional, and Caldwell, Harlan D., additional

Published
2013
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30. A live-attenuated chlamydial vaccine protects against trachoma in nonhuman primates

Author

Kari, Laszlo, primary, Whitmire, William M., additional, Olivares-Zavaleta, Norma, additional, Goheen, Morgan M., additional, Taylor, Lacey D., additional, Carlson, John H., additional, Sturdevant, Gail L., additional, Lu, Chunxue, additional, Bakios, Lauren E., additional, Randall, Linnell B., additional, Parnell, Michael J., additional, Zhong, Guangming, additional, and Caldwell, Harlan D., additional

Published
2011
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31. Frameshift Mutations in a Single Novel Virulence Factor Alter the In Vivo Pathogenicity of Chlamydia trachomatis for the Female Murine Genital Tract

Author

Sturdevant, Gail L., primary, Kari, Laszlo, additional, Gardner, Donald J., additional, Olivares-Zavaleta, Norma, additional, Randall, Linnell B., additional, Whitmire, William M., additional, Carlson, John H., additional, Goheen, Morgan M., additional, Selleck, Elizabeth M., additional, Martens, Craig, additional, and Caldwell, Harlan D., additional

Published
2010
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32. Chlamydia trachomatis Native Major Outer Membrane Protein Induces Partial Protection in Nonhuman Primates: Implication for a Trachoma Transmission-Blocking Vaccine

Author

Kari, Laszlo, primary, Whitmire, William M., additional, Crane, Deborah D., additional, Reveneau, Nathalie, additional, Carlson, John H., additional, Goheen, Morgan M., additional, Peterson, Ellena M., additional, Pal, Sukumar, additional, de la Maza, Luis M., additional, and Caldwell, Harlan D., additional

Published
2009
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33. The Chlamydia trachomatis Plasmid Is a Transcriptional Regulator of Chromosomal Genes and a Virulence Factor

Author

Carlson, John H., primary, Whitmire, William M., additional, Crane, Deborah D., additional, Wicke, Luke, additional, Virtaneva, Kimmo, additional, Sturdevant, Daniel E., additional, Kupko, John J., additional, Porcella, Stephen F., additional, Martinez-Orengo, Neysha, additional, Heinzen, Robert A., additional, Kari, Laszlo, additional, and Caldwell, Harlan D., additional

Published
2008
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34. Pathogenic Diversity amongChlamydia trachomatisOcular Strains in Nonhuman Primates Is Affected by Subtle Genomic Variations

Author

Kari, Laszlo, primary, Whitmire, William M., additional, Carlson, John H., additional, Crane, Deborah D., additional, Reveneau, Nathalie, additional, Nelson, David E., additional, Mabey, David C. W., additional, Bailey, Robin L., additional, Holland, Martin J., additional, McClarty, Grant, additional, and Caldwell, Harlan D., additional

Published
2008
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36. Chlamydia trachomatis virulence factor CT135 is stable in vivo but highly polymorphic in vitro.

Author

Bonner, Christine, Caldwell, Harlan D., Carlson, John H., Graham, Morag R., Kari, Laszlo, Sturdevant, Gail L., Tyler, Shaun, Zetner, Adrian, and McClarty, Grant

Subjects
CHLAMYDIA trachomatis, MICROBIAL virulence, GENETIC polymorphisms, PATHOGENIC microorganisms, BIOLOGICAL assay, SEXUALLY transmitted diseases
Abstract

Chlamydia trachomatis is an important human pathogen causing both ocular and sexually transmitted disease. Recently, we identified CT135 as an important virulence determinant in a mouse infection model. Results from CEL 1 digestion assays and sequencing analyses indicated that CT135 was much more polymorphic in high in vitro passage reference serovars than it was in clinical strains that had undergone limited passaging. Herein, we used targeted next-generation sequencing of the CT134-135 locus, from reference strains and clinical isolates, enabling accurate discovery of single nucleotide polymorphisms and other population genetic variations. Our results indicate that CT134 is stable in all C. trachomatis serovars examined. In contrast, CT135 is highly polymorphic in high-passaged reference ocular and non-LGV genital serovars, with the majority of the mutations resulting in gene disruption. In low-passaged ocular clinical isolates, CT135 was frequently disrupted, whereas in genital clinical isolates CT135 was intact in almost all instances. When a serovar K isolate, with an intact CT134 and CT135, was subjected to serial passage in vitro CT134 remained invariable, while numerous gene interrupting mutations rapidly accumulated in CT135. Collectively, our data indicate that, for genital serovars, CT135 is under strong positive selection in vivo, and negative selection in vitro. [ABSTRACT FROM AUTHOR]

Published
2015
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39. Classification and Prediction of Survival in Patients with the Leukemic Phase of Cutaneous T Cell Lymphoma

Author

Kari, Laszlo, primary, Loboda, Andrey, additional, Nebozhyn, Michael, additional, Rook, Alain H., additional, Vonderheid, Eric C., additional, Nichols, Calen, additional, Virok, Dezso, additional, Chang, Celia, additional, Horng, Wen-Hwai, additional, Johnston, James, additional, Wysocka, Maria, additional, Showe, Michael K., additional, and Showe, Louise C., additional

Published
2003
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40. Chlamydia pneumoniae Exacerbates Aortic Inflammatory Foci Caused by Murine Cytomegalovirus Infection in Normocholesterolemic Mice

Author

Burian, Katalin, primary, Berencsi, Klara, additional, Endresz, Valeria, additional, Gyulai, Zsofia, additional, Valyi-Nagy, Tibor, additional, Valyi-Nagy, Istvan, additional, Bakay, Marta, additional, Geng, Yuemei, additional, Virok, Dezso, additional, Kari, Laszlo, additional, Hajnal-Papp, Rozalia, additional, Trinchieri, Giorgio, additional, and Gonczol, Eva, additional

Published
2001
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41. Comprehensive global genome dynamics of Chlamydia trachomatisshow ancient diversification followed by contemporary mixing and recent lineage expansion

Author

Hadfield, James, Harris, Simon R., Seth-Smith, Helena M.B., Parmar, Surendra, Andersson, Patiyan, Giffard, Philip M., Schachter, Julius, Moncada, Jeanne, Ellison, Louise, Vaulet, María Lucíía Gallo, Fermepin, Marcelo Rodríííguez, Radebe, Frans, Mendoza, Suyapa, Ouburg, Sander, Morríííé, Servaas A., Sachse, Konrad, Puolakkainen, Mirja, Korhonen, Suvi J., Sonnex, Chris, Wiggins, Rebecca, Jalal, Hamid, Brunelli, Tamara, Casprini, Patrizia, Pitt, Rachel, Ison, Cathy, Savicheva, Alevtina, Shipitsyna, Elena, Hadad, Ronza, Kari, Laszlo, Burton, Matthew J., Mabey, David, Solomon, Anthony W., Lewis, David, Marsh, Peter, Unemo, Magnus, Clarke, Ian N., Parkhill, Julian, and Thomson, Nicholas R.

Abstract

Chlamydia trachomatisis the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis.

Published
2017
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43. Mutational Analysis of the Chlamydia muridarumPlasticity Zone

Author

Rajaram, Krithika, Giebel, Amanda M., Toh, Evelyn, Hu, Shuai, Newman, Jasmine H., Morrison, Sandra G., Kari, Laszlo, Morrison, Richard P., and Nelson, David E.

Abstract

ABSTRACTPathogenically diverse Chlamydiaspp. can have surprisingly similar genomes. Chlamydia trachomatisisolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarumshare 99% of their gene content. A region of high genomic diversity between Chlamydiaspp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarumcompared to C. trachomatisin the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarumPZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarumPZ genes are transcribed—and some may produce functional proteins—but are dispensable for infection of the murine genital tract.

Published
2015
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44. Antibody Signature of Spontaneous Clearance of Chlamydia trachomatis Ocular Infection and Partial Resistance against Re-challenge in a Nonhuman Primate Trachoma Model

Author

Kari, Laszlo, Bakios, Lauren E., Goheen, Morgan M., Bess, Leah N., Watkins, Heather S., Southern, Timothy R., Song, Lihua, Whitmire, William M., Olivares-Zavaleta, Norma, and Caldwell, Harlan D.

Subjects
*CHLAMYDIA trachomatis, *IMMUNOGLOBULINS, *SIGNATURE (Law), *TRACHOMA, *BLINDNESS, *DEVELOPMENTAL biology
Abstract

Background: Chlamydia trachomatis is the etiological agent of trachoma the world's leading cause of infectious blindness. Here, we investigate whether protracted clearance of a primary infection in nonhuman primates is attributable to antigenic variation or related to the maturation of the anti-chlamydial humoral immune response specific to chlamydial antigens. Methodology/Principal Findings: Genomic sequencing of organisms isolated throughout the protracted primary infection revealed that antigenic variation was not related to the inability of monkeys to efficiently resolve their infection. To explore the maturation of the humoral immune response as a possible reason for delayed clearance, sera were analyzed by radioimmunoprecipitation using intrinsically radio-labeled antigens prepared under non-denaturing conditions. Antibody recognition was restricted to the antigenically variable major outer membrane protein (MOMP) and a few antigenically conserved antigens. Recognition of MOMP occurred early post-infection and correlated with reduction in infectious ocular burdens but not with infection eradication. In contrast, antibody recognition of conserved antigens, identified as PmpD, Hsp60, CPAF and Pgp3, appeared late and correlated with infection eradication. Partial immunity to re-challenge was associated with a discernible antibody recall response against all antigens. Antibody recognition of PmpD and CPAF was destroyed by heat treatment while MOMP and Pgp3 were partially affected, indicating that antibody specific to conformational epitopes on these proteins may be important to protective immunity. Conclusions/Significance: Our findings suggest that delayed clearance of chlamydial infection in NHP is not the result of antigenic variation but rather a consequence of the gradual maturation of the C. trachomatis antigen-specific humoral immune response. However, we cannot conclude that antibodies specific for these proteins play the primary role in host protective immunity as they could be surrogate markers of T cell immunity. Collectively, our results argue that an efficacious subunit trachoma vaccine might require a combination of these antigens delivered in their native conformation. [ABSTRACT FROM AUTHOR]

Published
2013
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45. Phenotypic rescue of Chlamydia trachomatis growth in IFN-γ treated mouse cells by irradiated Chlamydia muridarum.

Author

Nelson, David E., Taylor, Lacey D., Shannon, Jeffrey G., Whitmire, William M., Crane, Deborah D., McClarty, Grant, Su, Hua, Kari, Laszlo, and Caldwell, Harlan D.

Subjects
CHLAMYDIA trachomatis, CHLAMYDIA infections, PATHOGENIC microorganisms, BACTERIAL toxins, BACTERIAL growth, TOXIN receptors, CELLULAR immunity, ANTIBODY-dependent cell cytotoxicity, MOUSE diseases
Abstract

Chlamydia trachomatis and C. muridarum, human and mouse pathogens, respectively, share more than 99% of open reading frames (ORFs) but differ in a cytotoxin locus. Presence or absence of cytotoxin gene(s) in these strains correlates with their ability to grow in IFN-γ treated mouse cells. Growth of toxin-positive C. muridarum is not affected in IFN-γ treated cells, whereas growth of toxin-negative C. trachomatis is inhibited. We previously reported that this difference in IFN-γ sensitivity is important to the in vivo infection tropism of these pathogens. Here we describe a phenotypic rescue assay that utilizes C. muridarum gamma irradiated killed elementary bodies (iEB) to rescue C. trachomatis infectivity in IFN-γ treated mouse cells. Rescue by iEB was temporal, maximal early post infection, directly related to multiplicity of iEB infection, and was independent of de novo chlamydial transcription. Lastly, C. muridarum iEB vacuoles and C. trachomatis inclusions were not fusogenic, suggesting the factor(s) responsible for rescue was secreted or exposed to the cytosol where it inactivated IFN-γ induced effectors. Chlamydial phenotypic rescue may have broader utility for the study of other EB associated virulence factors that function early in the interaction of chlamydiae with host cells. [ABSTRACT FROM AUTHOR]

Published
2007
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46. Chlamydia trachomatisPolymorphic Membrane Protein D Is a Virulence Factor Involved in Early Host-Cell Interactions

Author

Kari, Laszlo, Southern, Timothy R., Downey, Carey J., Watkins, Heather S., Randall, Linnell B., Taylor, Lacey D., Sturdevant, Gail L., Whitmire, William M., and Caldwell, Harlan D.

Abstract

ABSTRACTChlamydia trachomatisis an obligate intracellular mucosotropic pathogen of significant medical importance. It is the etiological agent of blinding trachoma and bacterial sexually transmitted diseases, infections that afflict hundreds of millions of people globally. The C. trachomatispolymorphic membrane protein D (PmpD) is a highly conserved autotransporter and the target of broadly cross-reactive neutralizing antibodies; however, its role in host-pathogen interactions is unknown. Here we employed a targeted reverse genetics approach to generate a pmpDnull mutant that was used to define the role of PmpD in the pathogenesis of chlamydial infection. We show that pmpDis not an essential chlamydial gene and the pmpDnull mutant has no detectable deficiency in cultured murine cells or in a murine mucosal infection model. Notably, however, the pmpDnull mutant was significantly attenuated for macaque eyes and cultured human cells. A reduction in pmpDnull infection of human endocervical cells was associated with a deficiency in chlamydial attachment to cells. Collectively, our results show that PmpD is a chlamydial virulence factor that functions in early host-cell interactions. This study is the first of its kind using reverse genetics to evaluate the contribution of a C. trachomatisgene to disease pathogenesis.

Published
2014
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47. Protocol for RNA fluorescence in situhybridization in mouse meningeal whole mounts

Author

Nilsson, Olof Rickard, Kari, Laszlo, Rosenke, Rebecca, and Steele-Mortimer, Olivia

Abstract

The multilayered meninges surrounding the brain and spinal cord harbor distinct immune cell populations with prominent roles in health and diseases. Here we present an optimized protocol for RNA fluorescence in situhybridization (RNA FISH) in meningeal whole mounts, allowing the visualization of gene expression. We also describe the combination of this protocol with immunohistochemistry for simultaneous visualization of mRNA and proteins. This protocol can be used for assessing spatial gene expression within the meninges.

Published
2022
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48. Chlamydia trachomatisPlasmid-Encoded Pgp4 Is a Transcriptional Regulator of Virulence-Associated Genes

Author

Song, Lihua, Carlson, John H., Whitmire, William M., Kari, Laszlo, Virtaneva, Kimmo, Sturdevant, Daniel E., Watkins, Heather, Zhou, Bing, Sturdevant, Gail L., Porcella, Stephen F., McClarty, Grant, and Caldwell, Harlan D.

Abstract

ABSTRACTChlamydia trachomatiscauses chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4knockout mutant exhibits an in vitrophenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.

Published
2013
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49. Frameshift Mutations in a Single Novel Virulence Factor Alter the In VivoPathogenicity of Chlamydia trachomatisfor the Female Murine Genital Tract

Author

Sturdevant, Gail L., Kari, Laszlo, Gardner, Donald J., Olivares-Zavaleta, Norma, Randall, Linnell B., Whitmire, William M., Carlson, John H., Goheen, Morgan M., Selleck, Elizabeth M., Martens, Craig, and Caldwell, Harlan D.

Abstract

ABSTRACTChlamydia trachomatisis 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. trachomatisinfection. 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 vitroprior 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 vitropassages. 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 vivovirulence characteristics, the strains exhibited no differences in the results of in vitroinfectivity 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.

Published
2010
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50. The Chlamydia trachomatisPlasmid Is a Transcriptional Regulator of Chromosomal Genes and a Virulence Factor

Author

Carlson, John H., Whitmire, William M., Crane, Deborah D., Wicke, Luke, Virtaneva, Kimmo, Sturdevant, Daniel E., Kupko, John J., Porcella, Stephen F., Martinez-Orengo, Neysha, Heinzen, Robert A., Kari, Laszlo, and Caldwell, Harlan D.

Abstract

ABSTRACTChlamydia trachomatispossesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless human C. trachomatisstrain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic of C. trachomatisstrains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 × 106inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 × 104IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes was glgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmid's ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.

Published
2008
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