Physiological Differences in C. neoformans in vitro versus in vivo and Their Effects on Antifungal

33 Pages·2016·0.89 MB·English

AAC Accepted Manuscript Posted Online 28 December 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.02108-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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2 Physiological Differences in C. neoformans in vitro versus in vivo and Their Effects on
3 Antifungal Susceptibility
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6 Nina T. Grossman,a Arturo Casadevalla1 n
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8 Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of f
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9 Public Health, Baltimore, MD 21205, USA.a

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12 Running Head: C. neoformans drug susceptibility in vitro vs in vivo m
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15 1Address correspondence to Arturo Casadevall, [email protected]. n
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24 ABSTRACT
25 Cryptococcus neoformans is an environmentally ubiquitous fungal pathogen that
26 primarily causes disease in people with compromised immune systems, particularly those with
27 advanced AIDS. There are estimated to be almost one million cases per year of cryptococcal
28 meningitis in patients with human immunodeficiency virus, leading to over 600,000 annual D
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29 deaths, with a particular burden in sub-Saharan Africa. Amphotericin B (AMB) and fluconazole n
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30 (FLC) are key components of cryptococcal meningitis treatment: AMB is used for induction, d
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31 FLC for consolidation, maintenance, and in occasional individuals, prophylaxis. However, the f
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32 results of standard antifungal susceptibility testing (AFST) for AMB and FLC do not correlate

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33 well with therapeutic outcomes, and, consequently, no clinical breakpoints have been
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34 established. While a number of explanations for this absence of correlation have been proffered, a
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35 one potential reason that has not been adequately explored is the possibility that the m
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36 physiological differences between the in vivo infection environment and the in vitro AFST r
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37 environment lead to disparate drug susceptibilities. These susceptibility-influencing factors n

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38 include melanization, which does not occur during AFST, the size of the polysaccharide capsule, n
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39 which is larger in infecting cells than in those grown under normal laboratory conditions, and the ry

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40 presence of large polyploid “titan cells,” which rarely occur in laboratory conditions. 2
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41 Understanding whether and how C. neoformans differentially expresses mechanisms of 9

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42 resistance to AMB and FLC in the AFST environment compared to the in vivo environment g
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43 could enhance our ability to interpret AFST results, and possibly lead to the development of s
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44 more applicable testing methods.
45
46 INTRODUCTION
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47 C. neoformans is a fungus responsible for causing lung infections that have the potential
48 to disseminate to the central nervous system (CNS) in the absence of adequate immune control,
49 and cause cryptococcal meningitis. This infection, which is most common among AIDS patients,
50 is uniformly fatal when left untreated. With antifungal treatment, 10-week fatality for AIDS
51 patients in developed countries is around 9%, while fatality in sub-Saharan Africa has been D
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52 estimated to be as high as 70% (1). In 2009, it was estimated that there were 958,000 HIV- n
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53 related cases per year worldwide, resulting in nearly 625,000 deaths, a disproportionate number d
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54 of which came from sub-Saharan Africa and South and South-east Asia (1). f
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55 Cryptococcal meningitis treatment occurs in three stages: induction, consolidation, and

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56 maintenance. Recommended induction therapy consists of amphotericin B (AMB) plus
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57 flucytosine, while fluconazole (FLC) is considered the optimal agent for the consolidation and a
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58 maintenance phases (2). FLC may also be used as an alternative agent for induction (2), a m
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59 substitution that is particularly common in resource-poor areas where AMB may be inaccessible r
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60 or difficult to administer (3). Given the high death rates of patients undergoing treatment for n

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61 cryptococcal meningitis, clinical practice would greatly benefit from the ability to predict those n
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62 drugs that an infection is likely to respond to, and at what doses. This would be particularly ry

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63 advantageous if it could be used to limit highly toxic and expensive AMB treatment to those 2
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64 patients unlikely to respond to FLC, thereby reducing costs, preventing unnecessary side effects, 9

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65 and preserving drug stocks in areas where availability is low. Many Candida species, for g
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66 example, have demonstrated that antifungal susceptibility testing (AFST) can be an effective tool s
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67 for predicting treatment outcome, and therefore guiding prescribing decisions. Based on updated,
68 species-specific breakpoints from CLSI, data compiled from a number of studies show that 92%
69 of fluconazole-susceptible Candida albicans, Candida tropicalis, and Candida parapsilosis were
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70 successfully treated with fluconazole, while the same was true for only 37% of resistant isolates
71 (4). Similar success has been achieved in correlating AFST results with treatment outcomes in
72 other azoles, such as voriconazole and itraconazole, as well (4). Unfortunately, no such
73 predictions have been adequately substantiated on the basis of standard AFST of C. neoformans.
74 Current guidelines from the Clinical and Laboratory Standards Institute (CLSI) D
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75 recommend testing the susceptibility of C. neoformans isolates by a microdilution method in n
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76 which suspensions of colonies are inoculated into RPMI 1640 broth containing increasing d
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77 dilutions of antifungal agent, incubating at 35°C for 70-74 hours, and visually determining the f
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78 minimum inhibitory concentration against an antifungal-free growth control well (5). However,

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79 several studies have failed to find any correlation between MICs from CLSI-recommended
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80 methods and treatment outcome for either AMB or FLC (6–10), and there is little evidence that a
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81 these standard assessments of susceptibility have clinical applicability (7, 11). Some research m
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82 suggests that different protocols may be more successful at predicting clinical outcomes for FLC r
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83 and AMB. In the case of FLC, a few studies have suggested that results from alternative n

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84 susceptibility testing methods, including microdilutions in yeast nitrogen base (YNB) broth and n
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85 E-tests, may have more clinical relevance (9, 10, 12, 13). For AMB, one group has had some ry

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86 success in predicting patient response using a method by which inoculum sizes are adjusted to 2
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87 reflect a patient’s fungal burden (8, 11). Nonetheless, the absence of a clear link between MICs 9

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88 from standard CLSI methodology and patient outcomes has undermined the efficacy of g
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89 performing AFST on C. neoformans, and made it impossible to establish clinical breakpoints s
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90 (14).
91 Multiple reasons have been hypothesized for the lack of clear correlation between
92 elevated antifungal MICs and cryptococcal meningitis treatment failure. Separate analyses of risk
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93 factors for FLC or AMB treatment failure in AIDS patients found that markers of high
94 cryptococcal burden were strong predictive factors (9, 15), implying that perhaps the effects of
95 varying fungal burden are masking less prominent effects of drug susceptibility (11).
96 Additionally, the finding that multiple C. neoformans strains are found in 18% of cryptococcosis
97 patients (16) has led to the suggestion that correlating MIC to treatment outcome may be D
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98 impeded by the concurrent presence of strains with different levels of susceptibility, of which n
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99 only one is tested (14). One study used a murine model to suggest that the pharmacodynamics of d
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100 FLC may prevent appropriate concentrations of drug from being reached and maintained in the f
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101 cerebrum (17). It has also been proposed that AMB may work in part through its positive

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102 immunomodulatory effects, which would not be reflected in an MIC (18).
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103 There is yet another potential explanation for the lack of correlation between FLC and a
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104 AMB MICs and treatment outcome that has not been thoroughly explored: that differences m
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105 between conditions in human tissues and in in vitro AFST result in fungal physiological states r
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106 that manifest large differences in susceptibility to FLC and AMB. This paper reviews a n

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107 significant literature showing various factors that have profound effects on C. neoformans n
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108 susceptibility to polyenes and azoles (Table 1). These factors include melanization, which does ry

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109 not occur in RPMI broth, the polysaccharide capsule, which is much larger in vivo than under 2
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110 normal in vitro conditions, the presence of large, polyploid “titan cells,” which grow in the host, 9

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111 but not in normal cultures, as well as a variety of expression changes that occur in the host that g
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112 have been hypothesized to affect ergosterol (Figure 1). A more thorough understanding of the s
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113 different compositional, morphological and transcriptional characteristics induced by the host
114 environment compared to AFST broth, and the extent to which they alter susceptibility to
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115 antifungal drugs, may help us interpret the results of AFST, or inform efforts to develop more
116 clinically relevant testing protocols.
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118 MELANIZATION
119 Among the many differences between in vitro AFST conditions and the host D
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120 environment, one of the most notable is the absence of phenolic substrates, such as L-DOPA. n
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121 These substrates, which are converted into melanin by C. neoformans laccase, were reported to d
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122 form black precipitate upon combination with antifungals in RPMI that prevents growth from f
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123 being assessed (19), thereby preventing them from being included in AFST broth. Melanin is a

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124 well-established virulence factor in C. neoformans that, in addition to protecting the yeast from a
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125 variety of environmental and host stresses, influences the susceptibility of many fungal species to a
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126 antifungal agents (18). There is abundant evidence that melanization increases C. neoformans m
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127 resistance to AMB, and some research suggests that it also affects FLC susceptibility. r
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128 C. neoformans melanizes in host tissues (20, 21), presumably by scavenging phenolic n

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129 precursors that are polymerized into melanin. Several studies have demonstrated that melanized n
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130 C. neoformans can withstand higher concentrations of AMB than non-melanized C. neoformans. ry

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131 Traditional AFST cannot detect differences between melanized and non-melanized cells, due to 2
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132 the inability of melanized cells inoculated into broth lacking phenolic substrates to produce 9

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133 melanized offspring, but time-kill assays can be used to assess the effects of melanization on g
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134 drug susceptibility (18). Time-kill assays have repeatedly shown that significantly more s
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135 melanized than non-melanized cells remain viable after incubation with various concentrations
136 of AMB (19, 22–24). This observation remains true whether cells are non-melanized due to
137 incubation without L-DOPA, or through laccase deficiency (19). Though the extent to which
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138 melanized cells out-survive non-melanized cells appears to differ by strain and drug
139 concentration, almost all studies showed at least a two-fold difference, with many assays
140 showing over six times the numbers of surviving melanized cells (19, 22–24). This suggests that
141 the practical effect of melanization on AMB susceptibility in vivo is likely to be considerable.
142 The mechanism by which melanin inhibits AMB was investigated using binding assays, D
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143 in which AMB was incubated with melanin. The melanin was then removed by centrifugation, n
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144 and the antifungal was used in antifungal susceptibility tests or killing assays, yielding much d
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145 higher MICs, and much less killing, from AMB incubated with melanin than not (19, 22). When f
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146 AMB was incubated with different doses of melanin, a dose-dependent relationship emerged in

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147 which higher doses of melanin particles led to higher AMB MICs (22). Additionally, one study
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148 conducted elemental analysis of melanin after incubation with AMB, and found a shift in C:N a
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149 ratios (19). These results led the authors of both studies to conclude that melanin increases C. m
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150 neoformans resistance by binding to AMB (19, 22), and in their review of the effects of r
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151 melanization in microbes, Nosanchuk and Casadevall hypothesized that this binding is likely to n

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152 inhibit AMB from reaching its targets (18). In addition, cell wall melanization results in a n
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153 reduction in its permeability as a result of smaller pore sizes (25, 26). Consequently, ry

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154 melanization may also block the entry of large molecules, such as AMB, by decreasing the 2
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155 porosity of the C. neoformans cell wall (18, 25, 26), in addition to its binding effects. Finally, it 9

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156 has also been suggested that melanin’s noted antioxidant properties (27–29) could protect C. g
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157 neoformans from the oxidative stress (30) that has been demonstrated to contribute to AMB’s s
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158 toxicity (30–32).
159 An inverse relationship between melanization and AMB susceptibility is suggested by in
160 vivo observations as well. In vitro laccase activity of C. neoformans strains isolated from cases of
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161 HIV-associated cryptococcal meningitis was found to be significantly negatively associated with
162 the rate of fungal clearance from human patients during AMB-based antifungal therapy (33).
163 Given that melanin is a virulence factor responsible for protection against phagocytosis and
164 killing by macrophages, among other host defenses (18), it is possible that the inverse
165 relationship between laccase activity and clearance occurred independently of any effects on D
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166 AMB activity. Nonetheless, these results are consistent with the notion that melanization may n
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167 increase the resistance of C. neoformans to AMB, and that in failing to account for the effects of d
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168 melanization, CLSI AFST is rendered less effective at accurately assessing a strain’s likelihood f
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169 of being cleared by AMB, as has been previously suggested (18, 19).

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170 Melanization was also demonstrated to reduce the efficacy of FLC, but the magnitude of
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171 this effect is unclear. A time-kill assay showed significantly higher survival of melanized cells a
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172 only in comparatively high concentrations of FLC (8 μg/ml) (22), while lower concentrations m
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173 had no significant effect (19, 22). However, as noted by van Duin et al., FLC is fungistatic, r
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174 rather than fungicidal, and showed little killing, suggesting that inhibitory effects of melanization n

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175 on FLC activity would be unlikely to be adequately demonstrated in an assay that is designed to n
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176 distinguish survival rates, rather than growth rates. The same binding assays as previously ry

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177 described for AMB showed no evidence of binding to FLC (19, 22), suggesting that the 2
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178 inhibitory effect of cryptococcal melanin on FLC activity is unlikely to act through the same 9

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179 mechanism as has been hypothesized for AMB. It is possible that melanin increases resistance to g
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180 FLC without binding the antifungal, perhaps by inhibiting its entry into the cell instead. s
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182 CAPSULE SIZE
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183 Another major difference between C. neoformans during infection compared to in vitro is
184 capsule size. The polysaccharide capsule is an important virulence factor with harmful effects on
185 the human immune system (34). Standard laboratory culture conditions, including growth on
186 RPMI, generally produce C. neoformans cells with relatively small capsules (35, 36). However,
187 cells from murine infections tend to have significantly larger capsules (37), and similar D
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188 phenomena have been shown in ex vivo human cerebrospinal fluid (CSF) (38). Furthermore, n
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189 there is no observed correspondence between the extent of an isolate’s capsule growth in vitro d
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190 and in vivo (34). The mechanisms by which capsule expansion inside the host is achieved have f
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191 yet to be completely elucidated, but several factors affect capsule size, including CO
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192 concentration (39), nutrient concentration (40), pH (40), iron availability (41), Mg2+ p
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193 concentration (42), certain phospholipids (43) and osmotic pressure (34). a
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194 Three separate studies have demonstrated that larger capsules protect C. neoformans from m
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195 AMB. These have induced large capsule growth comparable to that found in vivo by using low- r
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196 nutrient media (36), or by adding NaHCO to media, then incubating cells in 5% CO (44, 45). n
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197 Enlarged-capsule cells were then compared to normally grown small-capsule cells of the same n
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198 strain or strains using killing assays (36), time-kill curves (45), or CLSI broth microdilution ry

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199 methodology (44). The killing assays found significantly lower rates of killing of enlarged- 2
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200 capsule than small-capsule cells between AMB concentrations of 0.06 and 0.25 μg ml-1 after 9

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201 three hours (36). In time-kill curves performed on a set of clinical strains in 1 μg ml-1 of AMB, g
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202 100% and 75% of strains grown under capsule-enlarging conditions survived after 6 and 72 s
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203 hours, respectively, while only 35% and 0% of the same strains survived after the same
204 respective durations of exposure when grown normally (45). Finally, the geometric mean of
205 AMB MICs determined by broth microdilution for a collection of clinical isolates was
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206 significantly higher when the isolates were induced to grow large capsules (44). The protective
207 effect of capsule was further confirmed by one of the groups through the comparison of a wild-
208 type strain with an isogenic acapsular strain with a disrupted cap59 gene, and found that the
209 acapsular strain was more susceptible to AMB than the wild-type (36).
210 It is generally accepted that AMB’s primary mechanism of fungicidal activity is binding D
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211 to ergosterol, leading to the formation of pores in the fungal cell membrane. However, as n
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212 previously mentioned, AMB induces oxidative damage and intracellular accumulation of d
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213 reactive oxygen species (ROS) in several species of yeast, including C. neoformans (30–32). f
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214 Furthermore, by inhibiting ROS production, Candida tropicalis could be rendered less

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215 susceptible to AMB, suggesting that “ROS accumulation is a universal action mechanism of
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216 [AMB]” (32). In addition to testing the effects of capsule size on antifungal susceptibility, a
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217 Zaragoza et al. also demonstrated that C. neoformans cells with large capsules were protected m
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218 against killing by H O -induced ROS, and that isolated capsular polysaccharide could protect r
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219 cells with small capsules, likely by acting as an antioxidant. Therefore, it is probable that greater n

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220 production of capsular polysaccharide protects against AMB-induced ROS in the same manner, n
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221 and given the higher levels of capsule in vivo than in vitro, is likely to play a role in the limited ry

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222 efficacy of in vitro testing to assess in vivo AMB clearance (36). 2
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223 The role of capsule size in mediating FLC susceptibility is less clear. The E-test 9

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224 comparison of wild-type and acapsular strains found that the acapsular mutant was actually g
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225 highly resistant to FLC, and the authors theorized that the hydrophilic properties of both the s
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226 capsule and FLC could increase drug uptake into the cell (36). In contrast, the broth
227 microdilution found significantly higher FLC MICs among isolates with enlarged capsules,
228 compared to those grown normally (44). Interestingly, the two methods agreed on all other drugs
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