Cryptococcus neoformans Chitin Synthase 3 (Chs3) Plays a Critical Role in Dampening Host Inflammatory Responses

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan deficient C. neoformans lacking three chitin deacetylases (cda1Δ2Δ3Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase mediated formation of chitosan. Mice inoculated with chs3Δ at a dose previously shown to induce protection with cda1Δ2Δ3Δ die within 36 hours after installation of the organism. Mortality was not dependent on viable fungi as mice inoculated with heat-killed preparation of chs3Δ died at the same rate as mice inoculated with live chs3Δ, suggesting the rapid onset of death was host mediated likely caused by an over exuberant immune response. Histology, cytokine profiling, and flow cytometry indicates a massive neutrophil influx in the mice inoculated with chs3Δ. Mice depleted of neutrophils survived chs3Δ inoculation indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcal induced host inflammatory responses. IMPORTANCE Cryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host’s immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different than many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study we investigated the host responses to chs3Δ, a chitosan-deficient strain, and found mice inoculated with chs3Δ all died within 36 hours and death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

of patients with cryptococcal meningitis is between 10-30% in medically-advanced found to be protected against a subsequent challenge with wild-type KN99 (WT) C. 116 neoformans (10). Notably, this chitosan deficient strain is protective even when heat-killed 117 (10). Protective immunization is dependent on the inoculum size, as only mice that 118 received 10 7 CFU of cda1∆cda2∆cda3∆ were protectively immunized, mice that received 119 a lower inoculation were not protected (10). 120 Based on these data, we set out to test whether inoculation with other chitosan 121 deficient strains would also confer protection. We started this process using the new 122 chs3∆ strain which is chitosan deficient (Fig. 1C). We inoculated C57BL/6 mice 123 intranasally with 10 7 CFU of live cda1Δ2Δ3Δ (a concentration that is shown to be 124 protective for cda1∆2∆3∆), chs3∆, chs3∆::CHS3, or WT C. neoformans KN99 and were 125 monitored for survival. As expected, mice that received cda1Δ2Δ3Δ all survived the 126 infection and mice that received the WT KN99 or chs3∆::CHS3 all died or were euthanized Conversely, mice that received HK chs3∆ all died at the same rate as observed above 139 with live chs3∆ (Figs. 2 and 3A) indicating that mortality was not dependent on the viability 140 of the fungi. In addition, to confirm that the observed phenotype was due to loss of Chs3 141 and not some secondary mutation in the strain used, we assayed the original chs3∆ 142 mutant strain (Supplemental figure 1) and saw the same rapid mortality observed in 143 Figure 3A.

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A massive inflammatory response is triggered to chs3∆ inoculation. 155 The above data indicates that mice are not dying due to the fungal burden, as 156 death was not dependent on viable fungi in multiple mouse backgrounds (Figure 3). 157 These data suggest that the mortality associated with chs3∆ maybe host mediated (12). 158 To test this, C57BL/6 mice received an intranasal inoculation with 10 7 CFU HK WT KN99, 159 HK chs3∆ or HK chs3∆::CHS3 and the lungs were processed for histology. For all immune 160 studies, we chose to use heat-killed fungi to control for fungal burden as the WT KN99 and chs3∆::CHS3 would rapidly outgrow the chs3∆ strain and potentially skew our results. 162 Lungs were processed at 8 hours post inoculation as we could not constantly keep the    Discussion: 220 We have previously shown that deletion of a specific chitin synthase (CHS3), or 221 deletion of all three chitin deacetylases causes a significant reduction in chitosan in the 222 vegetative cell wall (9). These chitosan deficient strains of C. neoformans were found to 223 be avirulent and rapidly cleared from the murine lung (9). Moreover, infection with a 224 chitosan deficient C. neoformans strain lacking three chitin deacetylases (cda1Δ2Δ3Δ) 225 was found to confer protective immunity to a subsequent challenge with a virulent wild 226 type counterpart (10). These findings suggest that there is an altered host response to 227 chitosan-deficient strains. Surprisingly, we observed that mice inoculated with chitosan-  Depletion studies show a damaging role for neutrophils in the response to chs3Δ. 284 Altogether, chitosan plays a major role in the immune response to C. neoformans. In 285 addition, the response to chitosan deficient C. neoformans seems to depend on the type

Strain construction:
Gene-specific deletion construct of the chitin synthase 3 gene (CNAG_05581) was 297 generated using overlap PCR gene technology described previously (26,27) and 298 included the nourseothricin resistance cassette. The primers used to disrupt the genes 299 are shown in Table S1. The Chs3 deletion cassette contained the nourseothricin 300 resistance cassette resulting in a 1,539 bp replacement of the genomic sequence 301 between regions of primers 3-Chs3 and 6-Chs3 shown in upper case in Table S1. The 302 construct was introduced into the KN99a strain using biolistic techniques (28). To 303 generate a CHS3 complement strain, we replaced the NAT resistance cassette in 304 the chs3 deletion strain with the native CHS3 gene sequence by electroporation (29) (Table S2 for     Mice that lost 20% of the body weight at the time of inoculation or displayed signs of morbidity were considered ill and sacrificed. Data is representative of one experiment with 5 mice for KN99, 5 mice for cda1Δ2Δ3Δ, 10 mice for chs3∆, and 10 mice for chs3∆::CHS3. Virulence was determined using Mantel-Cox curve comparison with statistical significance determined by log-rank test. (***, P < 0.001). Data is cumulative of one experiment with 5 mice for KN99, and two experiments with 5 mice for chs3∆ and chs3∆::CHS3 each for a total of 10 mice. Virulence was determined using Mantel-Cox curve comparison with statistical significance determined by log-rank test. (***, P < 0.001).  Cytokine/chemokine responses were determined from the lung homogenates using the Bio-Plex Protein Array System. Data is cumulative of one experiment with 5 mice for PBS and KN99, and two experiments with 5 mice for chs3∆ and chs3∆::CHS3 each for a total of 10 mice experiments, ± standard errors of the means (SEM). Each dot represents data from an individual mouse. (***, P < 0.001) Figure 6. A significant increase in neutrophil recruitment in chs3∆ inoculated mice. C57BL/6 mice were inoculated with 10 7 Heat-killed CFUs of each strain by intranasal inoculation. At 8 hours post inoculation, pulmonary leukocytes were isolated from the lungs of each group and subjected to flow cytometry analysis (see Supplemental Table 2 for antibodies and Supplemental Fig. 3 for gating strategy). (A) total cell number of leukocytes. (B) Total and (C) percent neutrophils (CD11b + /CD24 + /Ly6G + /CD45 + ). Data is cumulative of one experiment with 5 mice for PBS and KN99, and two experiments with 5 mice for chs3∆ and chs3∆::CHS3 each for a total of 10 mice experiments, ± standard errors of the means (SEM). Each dot represents data from an individual mouse. (***, P < 0.001) Figure 7. Depletion of neutrophils protects chs3∆ inoculated mice: (A) C57BL/6, (B) BALB/c, or (C) CBA/J mice were inoculated with 10 7 Heat-killed CFUs of each strain by intranasal inoculation. Prior to inoculation and throughout the experiment, mice were treated with isotype antibody or anti-Ly6G antibody. Survival of the animals was recorded as mortality of mice for 20 days post inoculation. Mice that lost 20% of the body weight at the time of inoculation or displayed signs of morbidity were considered ill and sacrificed. Data is cumulative of two independent experiments with 5 mice for chs3∆ and chs3∆::CHS3 each for a total of 10 mice. Virulence was determined using Mantel-Cox curve comparison with statistical significance determined by log-rank test. (***, P < 0.001).
Supplemental figure 1: The original chs3∆ strain also induces rapid mortality: C57BL/6 mice were inoculated with 10 7 Heat-killed CFUs of each strain by intranasal inoculation. Survival of the animals was recorded as mortality of mice for 20 days post inoculation. Mice that lost 20% of the body weight at the time of inoculation or displayed signs of morbidity were considered ill and sacrificed. Data is representative of one experiment with 5 mice for each strain. Virulence was determined using Mantel-Cox curve comparison with statistical significance determined by log-rank test. (***, P < 0.001).
Supplemental figure 2. Cytokine/chemokine analysis: C57BL/6 mice were inoculated with 10 7 Heat-killed CFUs of each strain by intranasal inoculation. At 8 hours post inoculation, homogenates were prepared from the lungs of each group. Cytokine/chemokine responses were determined from the lung homogenates using the Bio-Plex Protein Array System. Data is cumulative of one experiment with 5 mice for PBS and KN99, and two experiments with 5 mice for chs3∆ and chs3∆::CHS3 each for a total of 10 mice experiments, ± standard errors of the means (SEM). Each dot represents data from an individual mouse.