Guanpingsheng Luo

Candida albicans Hyr1p Confers Resistance to Neutrophil Killing and Is a Potential Vaccine Target

Candida albicans is the most common cause of invasive fungal infections in humans. It is unclear how C. albicans escapes from phagocytic attack and survives in the hostile blood environment during life-threatening systemic infections. Using a conditional overexpression or suppression genetic strategy, we discovered that HYR1 gene reduced phagocytic killing activity of C. albicans in vitro and increased tissue fungal burden in vivo. Concordant with its positive regulation by the transcription factor Bcr1p, autonomous expression of HYR1 complemented the hypersusceptibility to phagocyte-mediated killing of a bcr1 null mutant of C. albicans in vitro. As for C. albicans, heterologous expression of HYR1 in Candida glabrata rendered the organism more resistant to neutrophil killing activity. Vaccination with a recombinant Hyr1p significantly protected mice against hematogenously disseminated candidiasis (P = .001). Finally, anti-rHyr1p polyclonal antibodies enhanced mouse neutrophil killing activity by directly neutralizing rHyr1p effects in vitro. Thus, Hyr1 is an important virulence factor for C. albicans, mediating resistance to phagocyte killing. Hyr1p is a promising target for vaccine or other immunological or small molecule intervention to improve the outcomes of disseminated candidiasis.

The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis.

Rhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first‐line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron‐depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross‐over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti‐Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti‐Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.

Gene overexpression/suppression analysis of candidate virulence factors of Candida albicans.

ABSTRACT We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors.

CotH3 mediates fungal invasion of host cells during mucormycosis

Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.

Isavuconazole Therapy Protects Immunosuppressed Mice from Mucormycosis

ABSTRACT We studied the in vitro and in vivo efficacies of the investigational drug isavuconazole against mucormycosis due to Rhizopus delemar. Isavuconazole was effective, with MIC and minimal fungicidal concentration (MFC) values ranging between 0.125 and 1.00 μg/ml. A high dose of isavuconazole prolonged the survival time and lowered the tissue fungal burden of cyclophosphamide/cortisone acetate-treated mice infected with R. delemar and was as effective as a high-dose liposomal amphotericin B treatment. These results support the further development of this azole against mucormycosis.

Combination Therapy of Murine Mucormycosis or Aspergillosis with Iron Chelation, Polyenes, and Echinocandins

ABSTRACT Liposomal amphotericin B (LAmB) combined wither either micafungin or deferasirox was synergistic in previous murine studies with mucormycosis or aspergillosis. We hypothesized that triple therapy using LAmB, micafungin, and deferasirox could further improve outcomes of mucormycosis or aspergillosis. Triple therapy improved survival and reduced tissue fungal burden of mice with mucormycosis and to a lesser extent with aspergillosis. Continued investigation into the use of triple therapy against mucormycosis and aspergillosis is warranted.

NDV-3 protects mice from vulvovaginal candidiasis through T- and B-cell immune response

We have previously reported that vaccination with rAls3p-N protein of Candida albicans, formulated with alum adjuvant (also designated as NDV-3) protects immunocompetent mice from, lethal disseminated candidiasis and mucosal oropharyngeal candidiasis. NDV-3 vaccine was recently, tested in a Phase 1 clinical trial and found to be safe, well-tolerated, and induced robust humoral and, cellular immune responses with increased interferon (IFN)-gamma and interleukin (IL)-17 secretion. In preparation for a Phase 2 clinical trial against vulvovaginal candidiasis (VVC), we evaluated NDV-3, efficacy in a murine VVC model. Here, NDV-3 induced a strong immune response characterized by high, anti-rAls3p-N serum IgG and vaginal IgA titers. Furthermore, moderate doses of the vaccine (a range of 1-30μg given subcutaneously [SQ] or 0.3-10μg given intramuscularly [IM]) elicited a 10-1000 fold, decrease in vaginal fungal burden vs. control (mice injected with alum adjuvant alone) in both inbred, and outbred mice infected with different clinical C. albicans isolates. Additionally, NDV-3 required both, T and B lymphocytes for efficacy in reducing C. albicans tissue burden, which is followed by a reduction, in neutrophil influx to the affected site. Finally, anti-rAls3p-N antibodies enhanced the ex vivo killing, of C. albicans by neutrophils primed with IFN-gamma. These data indicate that NDV-3 protects mice, from VVC by a mechanism that involves the concerted priming of both humoral and adaptive immune, responses.

Active and Passive Immunization with rHyr1p-N Protects Mice against Hematogenously Disseminated Candidiasis

We previously reported that Candida albicans cell surface protein Hyr1 encodes a phagocyte killing resistance factor and active vaccination with a recombinant N-terminus of Hyr1 protein (rHyr1p-N), significantly protects immunocompetent mice from disseminated candidiasis. Here we report the marked efficacy of rHyr1p-N vaccine on improving the survival and reducing the fungal burden of disseminated candidiasis in both immunocompetent and immunocompromised mice using the FDA-approved adjuvant, alum. Importantly, we also show that pooled rabbit anti-Hyr1p polyclonal antibodies raised against 8 different peptide regions of rHyr1p-N protected mice in a hematogenously disseminated candidiasis model, raising the possibility of developing a successful passive immunotherapy strategy to treat this disease. Our data suggest that the rabbit anti-Hyr1p antibodies directly neutralized the Hyr1p virulence function, rather than enhanced opsonophagocytosis for subsequent killing by neutrophil in vitro. Finally, the rHyr1p-N vaccine was protective against non-albicans Candida spp. These preclinical data demonstrate that rHyr1p-N is likely to be a novel target for developing both active and passive immunization strategies against Candida infections.

Efficacy of liposomal amphotericin B and posaconazole in intratracheal models of murine mucormycosis.

ABSTRACT Mucormycosis is a life-threatening fungal infection almost uniformly affecting diabetics in ketoacidosis or other forms of acidosis and/or immunocompromised patients. Inhalation of Mucorales spores provides the most common natural route of entry into the host. In this study, we developed an intratracheal instillation model of pulmonary mucormycosis that hematogenously disseminates into other organs using diabetic ketoacidotic (DKA) or cyclophosphamide-cortisone acetate-treated mice. Various degrees of lethality were achieved for the DKA or cyclophosphamide-cortisone acetate-treated mice when infected with different clinical isolates of Mucorales. In both DKA and cyclophosphamide-cortisone acetate models, liposomal amphotericin B (LAmB) or posaconazole (POS) treatments were effective in improving survival, reducing lungs and brain fungal burdens, and histologically resolving the infection compared with placebo. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies, and evaluate drug efficacies against life-threatening Mucorales infections.

Combination therapy with iron chelation and vancomycin in treating murine staphylococcemia

Iron acquisition is a virulence factor for Staphylococcus aureus. We assessed the efficacy of the iron chelator, deferasirox (Def), alone or in combination with vancomycin (Van) against two methicillin-resistant S. aureus (MRSA) strains in vitro and in a murine bacteremia model. In vitro time–kill assays were carried out against MRSA or vancomycin-intermediate S. aureus (VISA) strains. The impact of Def on Van binding to the surface of S. aureus was measured by flow cytometry. Furthermore, we compared the efficacy of Def, Van, or both drugs in treating S. aureus bacteremia in a murine model. Combination therapy reduced MRSA and VISA viability in vitro versus either drug alone or untreated controls (p < 0.005); this outcome was correlated with enhanced Van surface binding to S. aureus cells. In vivo, Def + Van combination therapy significantly reduced the bacterial burden in mice kidneys (p = 0.005) and spleen (p < 0.001), and reduced the severity of infection with MRSA or VISA strains compared to placebo-treated mice. Our results show that Def enhances the in vitro and in vivo capacity of Van-mediated MRSA killing via a mechanism that appears to involve increased binding of Van to the staphylococcal surface. Iron chelation is a promising, novel adjunctive therapeutic strategy for MRSA and VISA infections.