Browsing by Subject "Virulence"

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Although improvements in antifungal therapy have advanced the treatment of cryptococcosis, the mortality rate is approximately 12% in medically advanced countries, and approaches 50% in less developed regions. Additionally, C. neoformans can cause infection in seemingly healthy individuals, elevating its status as a primary human pathogen. Although numerous studies have examined virulence properties, less is understood regarding host immune factors in the lungs during early stages of fungal infection. In the present thesis studies, I examined the roles played by pulmonary surfactant proteins in response to C. neoformans in vitro and in vivo. We demonstrate that SP-D, but not SP-A, binds to the yeast and increases phagocytosis of poorly encapsulated yeast cells by macrophages, yet concomitantly protects the pathogenic microbes from macrophage-mediated defense mechanisms. Furthermore, we show that SP-D functions as risk factor in vivo by protecting the yeast cells against oxidant species and thus facilitating disease progression. The results of these studies provide a new paradigm on the role played by surfactant protein D during host responses to C. neoformans and, consequently, impart insight into potential future treatment strategies for cryptococcosis.

Cryptococcus neoformans is a human fungal pathogen that is also ubiquitous in the environment. To cause disease inside a human host, C. neoformans must be able to sense and respond to a multitude of stresses. One of the major responses to the host is the induction of a polysaccharide capsule, which allows the fungus to resist damage and evade the host immune response. This capsule is regulated by a number of signal transduction cascades, but a major contributor is the conserved cAMP/PKA pathway.

Using genetic and molecular biology techniques, I identified Gcn5 and Rim101 as key transcriptional regulators of capsule within the host. I determined that C. neoformans Rim101 is activated by a combination of the canonical pH sensing pathway and the cAMP/PKA pathway. This novel connection potentially gives the pathogen greater flexibility in responding to environmental stimuli, thus allowing for a greater capacity for disease.

I determined that the Rim101 transcription factor regulates cell wall remodeling in the context of the host by deep mRNA sequencing, electron microscopy, and biochemical assays. Using chromatin immunoprecipitation, I confirmed that these cell wall changes are under direct control of Rim101. I then confirmed the importance of cell wall changes in the host by nanoString profiling of fungal RNA in the context of a murine lung infection. I also examined the lungs of infected mice for cytokine and immune cell infiltrate and determined that C. neoformans cell wall changes are important in avoiding triggering an aberrant host response. I hypothesize that this cell wall remodeling via Rim101 activation is required for full capsule attachment and for masking immunogenic molecules from the host immune system.

Chlamydia trachomatis, a pathogen responsible for major diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to molecular genetic manipulation. The development of a system(s) for genetic analysis would significantly accelerate our ability to identify genes that enable Chlamydia to establish infection, survive within its host, and cause disease. This thesis describes two methods used to assess gene function in Chlamydia and to provide insights into its biology and pathogenesis. The first method described is based on specific inhibitors and is used to probe the role of lipooligosaccharide (LOS), a main lipid components of bacterial outer membranes. Using this approach, we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, blocks the synthesis of LOS in C. trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of anti-chlamydial agents.

The second part of this thesis describes the development of a system with which to perform forward genetics in C. trachomatis. Forward genetics approaches set out to identify the gene or set of genes that contributes to a specific biological process and usually entails generating random mutations in a large number of organisms, isolating mutants with an aberrant phenotype, and identifying the alleles associated with the mutant phenotype. In this approach, chemical mutagenesis is coupled with whole genome sequencing (WGS) and a system for DNA exchange within infected cells to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. We identified mutants with altered glycogen metabolism, including an attenuated strain defective for Type II secretion. The coupling of chemically induced gene variations and WGS to establish genotype-phenotype associations should be broadly applicable to the growing list of microorganisms intractable to traditional genetic mutational analysis.

Chlamydia trachomatis remains a highly relevant clinical pathogen as it is the causative agent of the most commonly reported sexually transmitted disease in the western hemisphere, and the most common cause of infectious blindness in the developing world. As an obligate intracellular pathogen, Chlamydia employs a vast assay of virulence proteins to hijack and remodel the host cellular machinery to facilitate its growth and dissemination. Besides delivering effector proteins into the host cytoplasm via a conserved type III secretion machinery, Chlamydia encodes components of multiple secretion systems, such as type II and IV. Chapter 3 of this document describes the secretion, processing and localization of two putative autotransporters (Pls1 and Pls2) and their involvement in inclusion expansion.

In recent years, many new chlamydial effector proteins have been described. CPAF (Chlamydial Protease-like Activity Factor) is a secreted serine protease that is emerging as a central virulence protein: it is proposed to play a central role in Chlamydia pathogenesis by cleaving proteins involved in antigen-presentation, apoptosis and cytoskeletal re-arrangements. However, the functional significance of CPAF remains elusive due to the lack of specific inhibitors and Chlamydia mutants. The body of work presented herein demonstrates that in addition to targeting host proteins, CPAF cleaves a subset of early chlamydial effector proteins, including Inc-proteins that reside on the nascent pathogenic vacuole ("inclusion"). The design and development of a CPAF-specific inhibitory peptide demonstrates that these chlamydial effector proteins are true targets of CPAF. This peptide reversed the cleavage of bacterial targets by CPAF both in an in vitro cleavage assay and during infection, indicating that these effectors are bona fide targets. Inhibition of CPAF activity also revealed that this protease regulates multiple facets of chlamydial pathogenesis. CPAF inhibition in infected epithelial cells led to the complete dismantling of the inclusion, secretion of pro-inflammatory cytokines and engagement of an inflammasome-dependent programmed cell death pathway. While fibroblasts defective in various inflammasome components were resistant to Chlamydia-induced cell death, inclusion integrity and bacterial replication was still compromised upon CPAF inhibition, indicating that loss of inclusion integrity was not a consequence of caspase-1 activation. Overall, these findings revealed that CPAF, in addition to regulating host function, directly modulates the activity of secreted effectors and early Inc-proteins. Furthermore, we establish that CPAF is an essential virulence factor that is required to maintain the integrity of the inclusion and prevent the engagement of innate immune programmed cell death pathways in infected epithelial cells. CPAF activity thus remains a compelling mechanism by which intracellular pathogens employ proteolytic events to modify the host environment.

Sexual development enables microbial pathogens to purge deleterious mutations from the genome and drives genetic diversity in the population. Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. C. neoformans has a defined a–α opposite sexual cycle (bisexual reproduction); however, >99% of clinical and environmental isolates are of the α mating type. Interestingly, α cells can undergo α–α unisexual reproduction, even involving genotypically identical cells. A central question is why would cells mate with themselves given that sex is costly and typically serves to admix pre-existing genetic diversity from genetically divergent parents? Sexual reproduction generates abundant spores that following inhalation, they penetrate deep into the alveoli of the lung, germinate, and establish a pulmonary infection growing as budding yeast. Therefore sex has been linked with virulence; however, hyphal development has been previously associated with reduced virulence and thus the roles of morphogenesis in virulence have not been extensively analyzed. To further understand the role of unisexual reproduction in C. neoformans we will investigate the evolutionary implications of α–α mating, explore its role in pathogenesis, and we will dissect the signaling pathway that regulates sexual development.

We isolated α–α unisexual reproduction progeny from the hyperfilamentous strain XL280 and subjected to a variety of phenotypic and genotypic assays (including whole genome sequencing and CGH). We found that unisexual and bisexual reproduction frequently generates phenotypic and genotypic diversity de novo, including aneuploidy. Aneuploidy was responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments, such as the mammalian host.

Interestingly aneuploidy strains that were fluconazole resistant were as virulent as the WT parental strain XL280. Although XL280 belongs to the serotype D lineage that exhibits limited pathogenicity, in further studies we found that is hypervirulent in the murine model. It can grow inside the lung of the host, establishing a pulmonary infection, and then disseminates to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is characterized by less protective immunity and is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies: 1) provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans (serotype D), and 2) reveal the virulence potential of serotype D that is broader and more dynamic than previously appreciated.

Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. To address this question we applied an insertional mutagenesis via Agrobacterium tumefaciens transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 governs cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. In further studies we found that Znf3 is required for sex- and mitotic-induced (SIS and MIS). SIS is less efficient in znf3 unilateral matings and is abolished in znf3 x znf3 bilateral matings, similar to the phenotypes of rdp1 mutants (the RNA-dependent RNA-polymerase of RNAi pathway). Znf3 is also required for transgene-induced mitotic silencing; znf3 mutations abrogate silencing of repetitive transgenes during vegetative growth. Znf3 tagged with mCherry is localized in the cytoplasm in bright, distinct foci. Co-localization of Znf3 with the P-body marker Dcp1-GFP further supports the hypothesis that Znf3 is a novel element of the RNAi pathway and operates to defend the genome during sexual development and vegetative growth. In concussion our studies provide further understanding of unisexual reproduction as an evolutionary successful strategy.