Tricia A. Missall

Mechanisms of Resistance to Oxidative and Nitrosative Stress: Implications for Fungal Survival in Mammalian Hosts

The ability of a fungal pathogen to cause disease requires the ability to survive in the host. Survival in the host is dependent on evasion of the hosts immune system, including the microbial killing mechanisms of phagocytes. The innate immune system is comprised of macrophages and neutrophils,

Distinct Stress Responses of Two Functional Laccases in Cryptococcus neoformans Are Revealed in the Absence of the Thiol-Specific Antioxidant Tsa1

ABSTRACT Laccases are thought to be important to the virulence of many fungal pathogens by producing melanin, a presumed oxygen radical scavenger. A laccase in Cryptococcus neoformans has been shown to synthesize melanin and contributes to the virulence and the survival in macrophages of this fungal pathogen. One C. neoformans laccase gene, LAC1, previously called CNLAC1, has been extensively studied, and we describe a homologous gene, LAC2, that is found 8 kb away from LAC1 in the genome. In this study we report a role for both laccases, in addition to the thiol peroxidase, Tsa1, in oxidative and nitrosative stress resistance mechanisms of C. neoformans. With use of real-time PCR, similar changes in expression of the two laccase genes occur in response to oxidative and nitrosative stresses, but only the regulation of the LAC2 gene during stress is influenced by Tsa1. Both laccases contribute to melanin production using L-dopa as a substrate and are differentially localized in the cell based on green fluorescent protein fusions. A single deletion of either LAC1 or LAC2 alone had no effect on sensitivity to H2O2 or nitric oxide. However, deletion of either LAC1 or LAC2 in combination with a TSA1 deletion resulted in a slight peroxide sensitivity, and a lac2Δ tsa1Δ deletion strain was sensitive to nitric oxide stress. In addition, the deletion of both laccases reduces survival of C. neoformans in primary macrophages. Based on our expression and functional analysis, we propose a novel model for the interaction of these two systems, which are both important for virulence.

Thiol peroxidase is critical for virulence and resistance to nitric oxide and peroxide in the fungal pathogen, Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen most commonly causing meningitis in immunocompromised patients. Current therapies are inadequate, and novel antifungal targets are needed. We have identified by proteomics two thiol peroxidases that are differentially expressed at 37°C, the temperature of the mammalian host. Consistent with their antioxidant role, we show that the genes encoding these thiol‐specific antioxidants, TSA1 and TSA3, are transcriptionally induced when C. neoformans is exposed to hydrogen peroxide. Genome sequence analysis of C. neoformans revealed a third thiol peroxidase, TSA4. We constructed single, double and triple mutants of the thiol peroxidase genes through homologous recombination and analysed their function by comparing the growth of these mutants with that of the wild‐type strain. The tsa1Δ mutant shows sensitivity to hydrogen peroxide and t‐butylhydroperoxide, as well as significant growth retardation at 25°C and 38.5°C. The tsa1Δ mutant is also sensitive to NO, demonstrating a link between oxidative and nitrosative stress pathways. In two mouse models of cryptococcosis, the tsa1Δ mutant is significantly less virulent.

Posttranslational, Translational, and Transcriptional Responses to Nitric Oxide Stress in Cryptococcus neoformans: Implications for Virulence

ABSTRACT The ability of the fungal pathogen Cryptococcus neoformans to evade the mammalian innate immune response and cause disease is partially due to its ability to respond to and survive nitrosative stress. In this study, we use proteomic and genomic approaches to elucidate the response of C. neoformans to nitric oxide stress. This nitrosative stress response involves both transcriptional, translational, and posttranslational regulation. Proteomic and genomic analyses reveal changes in expression of stress response genes. In addition, genes involved in cell wall organization, respiration, signal transduction, transport, transcriptional control, and metabolism show altered expression under nitrosative conditions. Posttranslational modifications of transaldolase (Tal1), aconitase (Aco1), and the thiol peroxidase, Tsa1, are regulated during nitrosative stress. One stress-related protein up-regulated in the presence of nitric oxide stress is glutathione reductase (Glr1). To further investigate its functional role during nitrosative stress, a deletion mutant was generated. We show that this glr1Δ mutant is sensitive to nitrosative stress and macrophage killing in addition to being avirulent in mice. These studies define the response to nitrosative stress in this important fungal pathogen.

Function of the thioredoxin proteins in Cryptococcus neoformans during stress or virulence and regulation by putative transcriptional modulators

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase and NADPH, is known to protect cells against oxidative stress. This disulphide reducing system is present in Cryptococcus neoformans and consists of two small, dithiol thioredoxin proteins and one thioredoxin reductase. In this study, we describe the thioredoxin proteins, Trx1 and Trx2, and present their importance not only to stress resistance, but also to the virulence of C. neoformans. Using real‐time polymerase chain reaction, we show the induction of both thioredoxin genes during oxidative and nitrosative stress. We describe through deletion studies that the trx1Δ mutant has a severe growth defect and is sensitive to multiple stresses, while the trx2Δ mutant is only sensitive to nitric oxide stress. Using gene replacement studies, we demonstrate that the thioredoxin protein products are partially redundant in function, but there is differential gene regulation which is especially important to nitrosative stress resistance. We have also identified two putative transcription factors, Atf1 and Yap4, which appear to differentially regulate the thioredoxin system under different conditions. Atf1 is necessary for oxidative stress induction, and Yap4 is necessary for nitrosative stress induction of the thioredoxin genes in C. neoformans. While these two putative transcription factors each appear to be dispensable for survival in macrophages and virulence in mice, we show the more highly expressed thioredoxin, TRX1, is necessary for survival of C. neoformans in the oxidative environment of macrophages and important for virulence of this fungal pathogen.

Thioredoxin Reductase Is Essential for Viability in the Fungal Pathogen Cryptococcus neoformans

ABSTRACT Thioredoxin reductase (TRR1) is an important component of the thioredoxin oxidative stress resistance pathway. Here we show that it is induced during oxidative and nitrosative stress and is preferentially localized to the mitochondria in Cryptococcus neoformans. The C. neoformans TRR1 gene encodes the low-molecular-weight isoform of the thioredoxin reductase enzyme, which shares little homology with that of its mammalian host. By replacing the endogenous TRR1 promoter with an inducible copper transporter promoter, we showed that Trr1 appears to be essential for viability of this pathogenic fungus, making it a potential antifungal target.

Immunohistochemical differentiation of four benign eccrine tumors

Background:  The histogenesis and differentiation of eccrine tumors, including cylindroma, poroma, spiradenoma and syringoma, remains controversial. This controversy may be because of sporadic and incomplete studies of these neoplasms.

Identification of Helicobacter pylori in skin biopsy of prurigo pigmentosa.

A 23-year-old Chinese man presented with a 3-year history of a pruritic eruption. On examination, pink urticarial papules associated with hyperpigmented reticulated patches were noted on his neck, back, and upper chest. Histopathology revealed vacuolar interface dermatitis and numerous gram-negative rods within a dilated hair follicle. The organisms were reactive with anti-Helicobacter pylori immunohistochemisty. The histologic findings and clinical presentation support the diagnosis of prurigo pigmentosa. Additional testing demonstrated a positive urease breath test and serum H. pylori IgG antibodies. The patient was referred to gastroenterology and treated with appropriate antibiotics. After treatment, esophagogastroduodenoscopy revealed chronic gastritis without evidence of H. pylori infection and his skin showed reticulated hyperpigmented patches without evidence of active inflammatory papules. Although previous reports have associated prurigo pigmentosa to H. Pylori gastritis, this is the first report of H. pylori organisms identified in a skin biopsy of prurigo pigmentosa.

Lentiginous melanoma in situ treatment with topical imiquimod: need for individualized regimens.

M elanoma in situ, lentiginous type (LM), is a precursor lesion for invasive malignant melanoma, lentiginous type (LMM). Already the most prevalent subtype of in situ melanoma, LM has been shown to be increasing in incidence. Currently, nonsurgical patients with LM have no treatment alternative but irradiation and so must endure the associated adverse effects of this treatment. In addition, recurrence following standard therapies is unacceptably high (8%-20%). For these reasons, a new effective therapy for LM that provides local control, prevents progression to LMM, and decreases morbidity and mortality is clinically desirable. Small studies have reported successful treatment of LM with imiquimod, 5%, cream. The present case series highlights 15 LM lesions in 14 patients treated with topical imiquimod. Histologic tissue specimens obtained before, during, and after treatment were evaluated to assist in directing patient management and in providing objective posttreatment histopathologic response.

The Use of Imiquimod to Minimize the Surgical Defect When Excising Invasive Malignant Melanoma Surrounded by Extensive Melanoma In Situ, Lentiginous Type

Malignant melanoma in situ, lentiginous type (LM), is a precursor lesion for malignant melanoma, lentiginous type (LMM). LM is characterized as an irregular, pigmented patch that is most commonly found on chronically sun-exposed skin, including the head and neck. LM is shown to be increasing in incidence and is the most prevalent subtype of in situ melanoma. Although LM constitutes only 4% to 15% of all melanomas, it is the most common type of head and neck melanoma. The progression of LM to its invasive form, LMM, is estimated to occur after 10 to 15 years. During this time, there is a protracted radial growth phase that is unique to LM among the in situ melanomas. Local recurrence of LMM is reported to account for 37% of all locally recurrent melanomas, which is most likely influenced by the clinical and histopathologic challenge of defining tumor margins in the context of chronically sun exposed skin. Although the recommended surgical margin for melanoma in situ is 5 mm, it has been well documented that, for the LM subtype, this is often inadequate and most likely contributes to local recurrence. The formation of LM, as well as its progression to LMM, is thought to involve the decrease in cell-mediated immunity in sun-damaged skin. Although first-line recommended treatment for LM is surgical excision, noninvasive therapies may be considered in certain clinical situations. Imiquimod is a topical immune-modulator acting on Toll-like receptors and has been shown to be a potent enhancer of innate and acquired immune responses. Various small open-label studies have reported treatment of LM with 5% imiquimod cream. The current study reports the use of this topical drug as an adjunct to surgical excision when treating extensive LM with focal invasive malignant melanoma.