CXCL1: A new diagnostic biomarker for human tuberculosis discovered using Diversity Outbred mice.

More humans have died of tuberculosis (TB) than any other infectious disease and millions still die each year. Experts advocate for blood-based, serum protein biomarkers to help diagnose TB, which afflicts millions of people in high-burden countries. However, the protein biomarker pipeline is small. Here, we used the Diversity Outbred (DO) mouse population to address this gap, identifying five protein biomarker candidates. One protein biomarker, serum CXCL1, met the World Health Organization's Targeted Product Profile for a triage test to diagnose active TB from latent M.tb infection (LTBI), non-TB lung disease, and normal sera in HIV-negative, adults from South Africa and Vietnam. To find the biomarker candidates, we quantified seven immune cytokines and four inflammatory proteins corresponding to highly expressed genes unique to progressor DO mice. Next, we applied statistical and machine learning methods to the data, i.e., 11 proteins in lungs from 453 infected and 29 non-infected mice. After searching all combinations of five algorithms and 239 protein subsets, validating, and testing the findings on independent data, two combinations accurately diagnosed progressor DO mice: Logistic Regression using MMP8; and Gradient Tree Boosting using a panel of 4: CXCL1, CXCL2, TNF, IL-10. Of those five protein biomarker candidates, two (MMP8 and CXCL1) were crucial for classifying DO mice; were above the limit of detection in most human serum samples; and had not been widely assessed for diagnostic performance in humans before. In patient sera, CXCL1 exceeded the triage diagnostic test criteria (>90% sensitivity; >70% specificity), while MMP8 did not. Using Area Under the Curve analyses, CXCL1 averaged 94.5% sensitivity and 88.8% specificity for active pulmonary TB (ATB) vs LTBI; 90.9% sensitivity and 71.4% specificity for ATB vs non-TB; and 100.0% sensitivity and 98.4% specificity for ATB vs normal sera. Our findings overall show that the DO mouse population can discover diagnostic-quality, serum protein biomarkers of human TB.

Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

Use of VHH antibodies for the development of antigen detection test for visceral leishmaniasis.

We have recently developed a sensitive and specific urine-based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li-isd1), tryparedoxin1 (Li-txn1) and nuclear transport factor 2 (Li-ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li-isd1 and Li-ntf2. This new assay was specific and had analytical sensitivity of 15-45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li-txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.

Proteomic Analysis of Urine from Patients with Plasmodium vivax Malaria Unravels a Unique Plasmodium vivax Protein That Is Absent from Plasmodium falciparum.

Five species of Plasmodium cause malaria in humans and two of them, P. vivax and P. falciparum, pose the greatest threat. Rapid antigen detection tests (RADT) have been used for many years to diagnose and distinguish malaria caused by these two parasites. P. falciparum malaria can single-handedly be diagnosed using an RADT, which detects the unique P. falciparum specific histidine-rich protein 2 (HRP2). Unfortunately, there is no RADT that can single-handedly diagnose P. vivax malaria because no specific marker of this parasite has yet been described. Here, we report the discovery of a unique P. vivax protein (Vir14, NCBI Reference Sequence: XP_001612449.1) that has no sequence similarity with proteins of P. falciparum and no significant similarities with proteins of other species of Plasmodium. We propose that this protein could be an outstanding candidate molecule for the development of a promising RADT that can single-handedly and specifically diagnose P. vivax malaria.

Assessment of a New Antigen Detection Test for the Diagnosis of Canine Visceral Leishmaniasis.

Canine visceral leishmaniasis (CVL) is a serious zoonotic disease in Brazil and Southern Europe. CVL is primarily caused by Leishmania infantum and its diagnosis relies largely on detection of parasites in bone marrow or lymph node aspirates by microscopic observation of the parasites in stained smears, parasite culture, or polymerase chain reaction (PCR). Serological tests exist but they do not distinguish active disease from simple exposure to parasite antigens. Here, we have assessed the utility of a new monoclonal antibody--based antigen (protein) detection test for the diagnosis of CVL. The test was positive in 70% of beagle dogs experimentally infected with L. infantum. In contrast, culture of the parasites from bone marrow aspirates was positive in only 40% of the infected animals. These preliminary results suggest that this antigen detection test, which we have recently described for the diagnosis of human VL, has the potential to be a useful diagnostic tool for CVL.

Urine-Based Antigen (Protein) Detection Test for the Diagnosis of Visceral Leishmaniasis.

This review describes and appraises a novel protein-based antigen detection test for visceral leishmaniasis (VL). The test detects in patient's urine six proteins from Leishmania infantum (chagasi) and Leishmania donovani, the etiological agents of VL. The gold standard test for VL is microscopic observation of the parasites in aspirates from spleen, liver, or bone marrow (and lymph node for dogs). Culture of the parasites or detection of their DNA in these aspirates are also commonly used. Serological tests are available but they cannot distinguish patients with active VL from either healthy subjects exposed to the parasites or from subjects who had a successful VL treatment. An antigen detection test based on the agglutination of anti-leishmania carbohydrates antibody coated latex beads has been described. However, the results obtained with this carbohydrate-based test have been conflicting. Using mass spectrometry, we discovered six L. infantum/L. donovani proteins excreted in the urine of VL patients and used them as markers for the development of a robust mAb-based antigen (protein) detection test. The test is assembled in a multiplexed format to simultaneously detect all six markers. Its initial clinical validation showed a sensitivity of 93% and specificity of 100% for VL diagnosis.

Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum / Leishmania donovani.

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use.

Novel Antigen Detection Assay to Monitor Therapeutic Efficacy of Visceral Leishmaniasis.

Visceral leishmaniasis (VL) diagnosis is routinely performed by invasive liver, spleen, bone marrow, or lymph node biopsies, followed by microscopic identification of the parasites. Conventional serological tests cannot distinguish active disease from asymptomatic VL or from cured infection. Here, we report the initial validation of an enzyme-linked immunosorbent assay (ELISA) assembled to detect the Leishmania infantum/donovani antigens iron superoxide dismutase 1 (Li-isd1), tryparedoxin 1 (Li-trx1), and nuclear transport factor 2 (Li-ntf2) as a tool to monitor therapeutic efficacy of VL. The assembled ELISA detected the antigens in the urine samples from seven VL patients before initiation of therapy. Importantly, the antigens were no longer detected in all patients after completion of the treatment. These preliminary observations point to a promising tool to follow treatment efficacy of VL.

Immunogenicity in dogs and protection against visceral leishmaniasis induced by a 14kDa Leishmania infantum recombinant polypeptide.

In areas were human visceral leishmaniasis (VL) is endemic, the domestic dog is the main parasite reservoir in the infectious cycle of Leishmania infantum. Development of prophylactic strategies to lower the parasite burden in dogs would reduce sand fly transmission thus lowering the incidence of zoonotic VL. Here we demonstrate that vaccination of dogs with a recombinant 14kDa polypeptide of L. infantum nuclear transport factor 2 (Li-ntf2) mixed with adjuvant BpMPLA-SE resulted in the production of specific anti-Li-ntf2 IgG antibodies as well as IFN-γ release by the animals' peripheral blood mononuclear cells stimulated with the antigen. In addition, immunization with this single and small 14kDa poplypeptide resulted in protracted progression of the infection of the animals after challenging with a high dose of virulent L. infantum. Five months after challenge the parasite load was lower in the bone marrow of immunized dogs compared to non-immunized animals. The antibody response to K39, a marker of active VL, at ten months after challenge was strong and significantly higher in the control dogs than in vaccinated animals. At the study termination vaccinated animals showed significantly more liver granulomas and lymphoid hyperplasia than non-vaccinated animals, which are both histological markers of resistance to infection. Together, these results indicate that the 14kDa polypeptide is an attractive protective molecule that can be easily incorporated in a leishmanial polyprotein vaccine candidate to augment/complement the overall protective efficacy of the final product.

Lung necrosis and neutrophils reflect common pathways of susceptibility to Mycobacterium tuberculosis in genetically diverse, immune-competent mice.

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis in susceptible humans. Here, we infected Diversity Outbred (DO) mice with ∼100 bacilli by aerosol to model responses in a highly heterogeneous population. Following infection, 'supersusceptible', 'susceptible' and 'resistant' phenotypes emerged. TB disease (reduced survival, weight loss, high bacterial load) correlated strongly with neutrophils, neutrophil chemokines, tumor necrosis factor (TNF) and cell death. By contrast, immune cytokines were weak correlates of disease. We next applied statistical and machine learning approaches to our dataset of cytokines and chemokines from lungs and blood. Six molecules from the lung: TNF, CXCL1, CXCL2, CXCL5, interferon-γ (IFN-γ), interleukin 12 (IL-12); and two molecules from blood - IL-2 and TNF - were identified as being important by applying both statistical and machine learning methods. Using molecular features to generate tree classifiers, CXCL1, CXCL2 and CXCL5 distinguished four classes (supersusceptible, susceptible, resistant and non-infected) from each other with approximately 77% accuracy using completely independent experimental data. By contrast, models based on other molecules were less accurate. Low to no IFN-γ, IL-12, IL-2 and IL-10 successfully discriminated non-infected mice from infected mice but failed to discriminate disease status amongst supersusceptible, susceptible and resistant M.-tuberculosis-infected DO mice. Additional analyses identified CXCL1 as a promising peripheral biomarker of disease and of CXCL1 production in the lungs. From these results, we conclude that: (1) DO mice respond variably to M. tuberculosis infection and will be useful to identify pathways involving necrosis and neutrophils; (2) data from DO mice is suited for machine learning methods to build, validate and test models with independent data based solely on molecular biomarkers; (3) low levels of immunological cytokines best indicate a lack of exposure to M. tuberculosis but cannot distinguish infection from disease.

Identification and diagnostic utility of Leishmania infantum proteins found in urine samples from patients with visceral leishmaniasis.

Despite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identify Leishmania infantum antigens produced in vivo in humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinct L. infantum proteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantum iron superoxide dismutase, NCBI accession number XP_001467866.1; L. infantum tryparedoxin, NCBI accession number XP_001466642.1; and L. infantum nuclear transport factor 2, NCBI accession number XP_001463738.1) were cloned, and the recombinant molecules were produced in Escherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect these L. infantum antigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based on L. infantum proteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.

Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases.

Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (K(D) near 1 nM) and include potent inhibitors of BoNT/A protease activity (K(i) near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.

Gnotobiotic piglet infection model for evaluating the safe use of antibiotics against Escherichia coli O157:H7 infection.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli (STEC), especially O157:H7, cause bloody diarrhea, and in 3%-15% of individuals the infection leads to hemolytic uremic syndrome (HUS) or other complications. Use of antibiotics to treat STEC infections is controversial. Here, we describe the use of piglets to evaluate the efficacy and mechanism of action of antibiotics in these infections. METHODS: The effects of 2 antibiotics on STEC toxin production and their mechanisms of action were first determined by enzyme-linked immunosorbent assay and subsequently evaluated clinically in the gnotobiotic piglet infection model. RESULTS: In vitro treatment of clinical and isogenic strains with ciprofloxacin increased the production of Stx2 via phage induction but not the production of Stx1. Azithromycin caused no significant increase in toxin production. After treatment with ciprofloxacin, infected piglets had diarrhea and the severe fatal neurological symptoms associated with Stx2 intoxication. Characteristic petechial hemorrhages in the cerebellum were more severe in ciprofloxacin-treated animals than in control animals. In contrast, azithromycin-treated piglets survived the infection and had little or no brain hemorrhaging. CONCLUSIONS: The increased in vitro toxin production caused by ciprofloxacin was strongly correlated with death and an increased rate of cerebellar hemorrhage, in contrast to the effect of azithromycin. The piglet is a suitable model for determining the effectiveness and safety of antibiotics available to treat patients.

Phagocytosis by human neutrophils is stimulated by a unique fungal cell wall component.

Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.

The Kluyveromyces lactis alpha1,6-mannosyltransferase KlOch1p is required for cell-wall organization and proper functioning of the secretory pathway.

Mutants of Kluyveromyces lactis denominated vga (vanadate glycosylation affected) bear various combinations of glycosylation and cell-wall defects. The vga3 mutation of K. lactis was mapped in the KlOCH1 gene, encoding the functional homologue of the Saccharomyces cerevisiaealpha1,6-mannosyltransferase. Quantitative analysis of cell-wall components indicated a noticeable increase of chitin and beta1,6-glucans and a severe decrease of mannoproteins in the mutant cells as compared with the wild-type counterparts. Fine-structure determination of the beta1,6-glucan polymer indicated that, in the vga3-1 strain, the beta1,6-glucans are shorter and have more branches than in the wild-type strain. This suggests that cell-wall remodelling changes take place in K. lactis in the presence of glycosylation defects. Moreover, the vga3 cells showed a significantly improved capability of secreting heterologous proteins. Such a capability, accompanied by the highly reduced N-glycosylation, may be of biotechnological interest, especially when hyper-glycosylation of recombinant products must be avoided.

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae.

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.

A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.

The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis, glycosylation, and cell wall properties.

Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen Candida albicans. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by GDA1, to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the C. albicans homologue of the Saccharomyces cerevisiae GDA1 gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the C. albicans disrupted gda1/gda1 strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The gda1/gda1 mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by beta-1,3-glucanases. Our results show that the C. albicans organism contains beta-mannose at their nonreducing end, differing from S. cerevisiae, which has only alpha-linked mannose residues in its O-glycans. Mutants lacking both alleles of GDA1 grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and N-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition. Gda1/gda1 cells offer a model for discriminating among them.

A Saccharomyces cerevisiae mutant with increased virulence.

Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.