Isolation and Characterization of Senescent Cryptococcus neoformans and Implications for Phenotypic Switching and Pathogenesis in Chronic Cryptococcosis

Although several virulence factors and associated genes have been identified, the mechanisms that allow Cryptococcus neoformans to adapt during chronic infection and to persist in immunocompromised hosts remain poorly understood. Characterization of senescent cells of C. neoformans demonstrated that these cells exhibit a significantly enlarged cell body and capsule but still cross the blood-brain barrier. C. neoformans cells with advanced generational age are also more resistant to phagocytosis and killing by antifungals, which could promote their selection during chronic disease in humans. Senescent cells of RC-2, a C. neoformans strain that undergoes phenotypic switching, manifest switching rates up to 11-fold higher than those of younger cells. Infection experiments with labeled cells suggest that senescent yeast cells can potentially accumulate in vivo. Mathematical modeling incorporating different switching rates demonstrates how increased switching rates promote the emergence of hypervirulent mucoid variants during chronic infection. Our findings introduce the intriguing concept that senescence in eukaryotic pathogens could be a mechanism of microevolution that may promote pathoadaptation and facilitate evasion of an evolving immune response.Cryptococcus neoformans is an encapsulated yeast that causes chronic meningoencephalitis predominantly in patients with advanced human immunodeficiency virus infection. Worldwide, this disease is estimated to cause more than 600,000 deaths per year (22). This high death rate may result in part from the fact that this chronic infection is notoriously difficult to eradicate, despite effective antifungal therapy. Even in successfully treated patients with access to antiretroviral therapy, the organism persists and can cause relapse both before and after the start of highly active antiretroviral treatment.In murine infection models, C. neoformans strain RC-2 can augment virulence by undergoing phenotypic switching from a smooth (SM) parent colony to a more virulent mucoid (MC) colony variant (6). Phenotypic switching occurs in both species and varieties of this fungus (6, 8, 11). Although differentially regulated genes associated with switching have been described (10), the precise molecular mechanism that controls phenotypic switching in C. neoformans is unknown. In vitro investigations have demonstrated that phenotypic switching occurs at a stable rate and MC switch variants spontaneously arise at a rate of 1 in about 20,000 plated SM colonies. Environmental signals that induce or alter this process have not been identified to date. Phenotypic switching occurs during chronic murine infection and alters the outcome (6), but it is noteworthy that MC switch variants consistently emerge late in the course of infection (after 6 weeks), although the fungal burden is highest on day 14 after intratracheal (i.t.) infection. This could reflect altering selection pressure by an evolving host response or, alternatively, a change in the microbe''s propensity to undergo phenotypic switching over time. In Candida albicans, the process of phenotypic switching is controlled by pathways that are involved in silencing and maintenance of genomic stability (14, 20, 34, 35). Furthermore, in the context of research on aging in Saccharomyces cerevisiae, it has become evident that senescent yeast cells (old cells with advanced generational age) exhibit less genomic stability (17, 18). C. neoformans is also a unicellular haploid yeast that replicates clonally in vivo. Consequently, we reasoned that it was conceivable that replicative aging of C. neoformans during chronic infection could alter genomic stability and its propensity to undergo phenotypic switching. We therefore investigated the effect of generational aging on phenotypic switching in C. neoformans and also compared it to C. albicans.     

Phenotypic Switching of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis><Emphasis Type="Italic">gattii</Emphasis>

Microorganisms that live in fluctuating environments must constantly adapt their behavior to survive. The host constitutes an important microenvironment in opportunistic and primary fungal pathogens like Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). In clonal populations, adaptation may be achieved through the generation of diversity. For fungi phenotype switching constitutes a mechanism that allows them to change rapidly. Both C. neoformans and C. gattii undergo phenotypic switching, which allows them to be successful pathogens and cause persistent disease. Similar to other encapsulated microbes that exhibit phenotypic variation, phenotypic switching in Cryptococcus changes the polysaccharide capsule. Most importantly, in animal models phenotypic switching affects virulence and can change the outcome of infection. Virulence changes because C. neoformans and C. gattii switch variants elicit different inflammatory responses in the host. This altered host response can also affect the response to antifungal therapy and in some cases may even promote the selection of switch variants. This review highlights the similarity and differences between phenotypic switching in C. neoformans and C. gattii, the two dominant species that cause cryptococcosis in humans.     

Phenotypic switching in Cryptococcus neoformans

Cryptococcus neoformans strains exhibit considerable phenotype variability with regards to the capsular polysaccharide, sterol composition of the cell wall, and cell and colony morphology. Phenotypic changes can occur spontaneously during in vitro passage of strains or during chronic infection in vivo and may be associated with differences in virulence. Studies from our laboratory have demonstrated that phenotype variability can be the result of phenotypic switching. Phenotypic switching is defined as a reversible change of an observable colony phenotype that occurs at a frequency above the expected frequency for somatic mutations. This implies that phenotypic switching represents controlled and programmed changes in this pathogenic yeast rather than random mutations. We have shown that a phenotypic switch from a smooth colony phenotype to a mucoid colony phenotype occurs in vitro and in vivo during chronic infection of mice. More importantly we have now demonstrated that the switch is associated with an increase in virulence and a change in the host immune response. Implications of these findings for the pathogenesis of cryptococcosis are discussed.     

Characterization of Phenotypic Switching in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Biofilms

Cryptococcus neoformans is an encapsulated yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients and also occasionally causes disease in apparently healthy individuals. This fungus collectively forms biofilms on polystyrene plates and medical devices, whereas individually can undergo phenotypic switching. Both events have profound consequences in the establishment of fungal infection and are associated with persistent infection due to increase resistance to antimicrobial therapy. In this study, we characterized switch phenotypes in C. neoformans biofilms. Smooth, mucoid, and wrinkled switch phenotypes of various switching C. neoformans strains were examined for their adhering and biofilm-forming ability on 96-well plates using cell counts and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, respectively. Both assays showed that C. neoformans strains with the parent smooth phenotype adhered and formed stronger biofilms than their mucoid and wrinkled counterparts. Furthermore, the phenotypic switching frequencies of the individual colony types grown in biofilms or as planktonic cells were investigated. For the parent smooth variant of most strains, we found enhanced phenotypic switching in cryptococcal biofilms when compared to switching rates of planktonic cells. In contrast, the back-switching rate of mucoid to smooth variant was significantly higher in planktonic cells of seven strains of C. neoformans strains. These results suggested that phenotypic switching can occur in cryptococcal biofilms and extend our understanding of the relationship of both phenomena.     

Gene Expression Switching of Receptor Subunits in Human Brain Development

Synaptic receptors in the human brain consist of multiple protein subunits, many of which have multiple variants, coded by different genes, and are differentially expressed across brain regions and developmental stages. The brain can tune the electrophysiological properties of synapses to regulate plasticity and information processing by switching from one protein variant to another. Such condition-dependent variant switch during development has been demonstrated in several neurotransmitter systems including NMDA and GABA. Here we systematically detect pairs of receptor-subunit variants that switch during the lifetime of the human brain by analyzing postmortem expression data collected in a population of donors at various ages and brain regions measured using microarray and RNA-seq. To further detect variant pairs that co-vary across subjects, we present a method to quantify age-corrected expression correlation in face of strong temporal trends. This is achieved by computing the correlations in the residual expression beyond a cubic-spline model of the population temporal trend, and can be seen as a nonlinear version of partial correlations. Using these methods, we detect multiple new pairs of context dependent variants. For instance, we find a switch from GLRA2 to GLRA3 that differs from the known switch in the rat. We also detect an early switch from HTR1A to HTR5A whose trends are negatively correlated and find that their age-corrected expression is strongly positively correlated. Finally, we observe that GRIN2B switch to GRIN2A occurs mostly during embryonic development, presumably earlier than observed in rodents. These results provide a systematic map of developmental switching in the neurotransmitter systems of the human brain.     

Evolution of CCR5 use before and during coreceptor switching

The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.     

Homologous chromosome recombination generating immunoglobulin allotype and isotype switch variants.

We investigated whether spontaneous isotype switching in monoclonal antibody-producing hybridomas always occurs with genes on the same chromosome. Spleen cells of (BAB/ 25 X AKR/J) F1 mice, immunized with dansyl-keyhole limpet hemocyanin (DNS-KLH), were hybridized with NS-1 to generate hybridomas producing monoclonal anti-DNS antibodies of either the b or d haplotype of the BAB/25 or AKR/J parent, respectively. We selected isotype switch variants of such hybridomas using the fluorescence-activated cell sorter (FACS). Although in most cases the allotypic haplotype expressed by the parent and switch-variant hybridomas are the same, in one family of variants we noted a switch in haplotype along with the switch in isotype. This was noted in the selection of IgG2a switch variants from an IgG1 switch variant originally derived from an IgG3-producing parent. Biochemical and molecular studies confirm that the allotype switch variant expresses the same heavy-chain variable region gene complex as its parent hybridomas. As such, the allotype switch represents an example of spontaneous mitotic recombination between immunoglobulin heavy-chain genes, generating a single actively transcribed gene from loci previously positioned on different chromosomes.     

Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching

The natural evolution of human immunodeficiency virus type 1 infection often includes a switch in coreceptor preference late in infection from CCR5 to CXCR4, a change associated with expanded target cell range and worsened clinical prognosis. Why coreceptor switching takes so long is puzzling, since it requires as few as one to two mutations. Here we report three obstacles that impede the CCR5-to-CXCR4 switch. Coreceptor switch variants were selected by target cell replacement in vitro. Most switch variants showed diminished replication compared to their parental R5 isolate. Transitional intermediates were more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 virus or the final R5X4 (or rare X4) variant. The small number of mutations in viruses selected for CXCR4 use were distinctly nonrandom, with a dominance of charged amino acid substitutions encoded by G-to-A transitions, changes in N-linked glycosylation sites, and isolate-specific mutation patterns. Diminished replication fitness, less-efficient coreceptor use, and unique mutational pathways may explain the long delay from primary infection until the emergence of CXCR4-using viruses.     

Genetic analysis of in vivo-selected viral variants causing chronic infection: importance of mutation in the L RNA segment of lymphocytic choriomeningitis virus.

Viral variants with different biological properties are present in the central nervous systems (CNS) and lymphoid tissues of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Viral isolates from the CNS are similar to the original Armstrong LCMV strain and induce potent virus-specific T-cell responses in adult mice, and the infection is rapidly cleared. In contrast, LCMV isolates derived from spleens of carrier mice cause persistent infections in adult mice. This chronic infection is associated with low levels of antiviral T-cell responses. In this study, we genetically characterized two independently derived spleen variants by making recombinants (reassortants) between the spleen isolates and wild-type (wt) LCMV and showed that the ability to persist in adult mice and the associated suppression of T-cell responses segregates with the large (L) RNA segment. In addition, we analyzed a revertant (isolated from the CNS) derived from one of the spleen variants. By comparing the biological properties of three reassortants that contained the same S segment but had the L segment of either the original wt Armstrong LCMV, the spleen variant derived from it, or the CNS revertant derived from the spleen variant, we were able to show unequivocally that biologically relevant mutations occurred in the L segment not only during generation of the spleen variant from wt LCMV but also in reversion of the spleen variant to the wt phenotype. Thus, our results showed that (i) genetic alterations in the L genomic segment were involved in organ-specific selection of viral variants, and (ii) these mutations profoundly affected the ability of LCMV to cause chronic infections in adult mice.     

Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.     

High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching

Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high-frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.     

Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence.

Strains of the murine coronavirus mouse hepatitis virus type 4 (MHV-4) which contained a mutation in the E2 peplomer glycoprotein were obtained by selection for resistance to neutralization by monoclonal antibodies. Characterization of six variants representing two independent epitopes on E2, E2B and E2C, by in vitro neutralization and antibody-binding assays demonstrated that selection for an alteration in epitope E2B also resulted in changes in epitope E2C and vice versa. We observed a mutation frequency of approximately 10(-4.3) to 10(-4.6), which is consistent with the expected occurrence of single point mutations. The variant virus strains were attenuated with respect to neurovirulence when compared with wild-type MHV-4. Mice normally develop encephalomyelitis and die after wild-type MHV-4 infection. Mice receiving 2- to 3-log-higher doses of the variant strains survived and developed demyelinating disease. As the disease progressed, evidence of remyelination and ongoing demyelination was observed up to 65 days after infection. Virus reisolated 15 days after infection retained the variant phenotype. The data indicate that the E2 glycoprotein plays a central role in determining the cellular tropism and virulence of MHV-4 in the mouse.     

Fungal mating: Candida albicans flips a switch to get in the mood

The fungal pathogen Candida albicans can mate under highly controlled conditions. It can also undergo phenotypic switching. A recent discovery joins these disparate processes to reveal that 'opaque' switch variants mate 10(6) times better than 'white' variants.     

Isolation and characterization of a protective bacterial culture isolated from honey active against American Foulbrood disease

The cell surface of mycobacteria is quite rich in lipids. Glycopeptidolipids, surface-exposed lipids that typify some mycobacterial species, have been associated with a phenotypic switch between rough and smooth colony morphotypes. This conversion in Mycobacterium smegmatis is correlated with the absence/presence of glycopeptidolipids on the cell surface and is due to insertion sequence mobility. Here, we show that the occurrence of a high amount of glycopeptidolipids in the smooth variant leads to lower invasion abilities and lower internalization by macrophages. We further show that the high production of glycopeptidolipids on the cell surface can confer a selective advantage to the smooth variant when grown in vitro . This higher fitness under the laboratory condition might explain the selection of smooth variants in several independent laboratories. The implications of these findings are discussed.     

In Vitro Selection of Variants of Herpes Simplex Virus Type 1 Which Differ in Cytopathic Changes

To analyze the mechanisms for in vitro emergence of the syncytial variants of herpes simplex virus type 1 (HSV-1), several cell lines were infected with a mixture of equal amounts of two HSV-1 variants, one syncytial and the other non-syncytial, and changes in their relative abundance were monitored during passage. With a combination of two variants of the Miyama strain of HSV-1, the syncytial variant became dominant during passage in Vero, RK-13 and FL cells. On the other hand, the ratios of the two variants remained around 1:1 during the passage in HEp-2, MGC and HEL cells. In another set of variants of the SKO strain of HSV-1, the outcomes were different from those of the Miyama strain in the FL, MGC and HEp-2 cells. The ratios of the two variants remained around 1:1 during passage in FL cells, while the non-syncytial variant became dominant during passage in MGC and HEp-2 cells. In addition, we examined the effects of a complement and interferon-β (IFN-β) on the outcome of the selection. As a result, the complement slowed the selection of a syncytial variant, whereas IFN-β facilitated it.     

In vivo selection of lymphocyte-tropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection.

This study demonstrates cell-specific selection of viral variants during persistent lymphocytic choriomeningitis virus infection in its natural host. We have analyzed viral isolates obtained from CD4+ T cells and macrophages of congenitally infected carrier mice and found that three types of variants are present in individual carrier mice: (i) macrophage-tropic, (ii) lymphotropic, and (iii) amphotropic. The majority of the isolates were amphotropic and exhibited enhanced growth in both lymphocytes and macrophages. However, some of the lymphocyte-derived isolates grew well in lymphocytes but poorly in macrophages, and a macrophage-derived isolate replicated well in macrophages but not in lymphocytes. In striking contrast, the original wild-type (wt) Armstrong strain of lymphocytic choriomeningitis virus that was used to initiate the chronic infection and from which the variants are derived grew poorly in both lymphocytes and macrophages. These three types of variants also differed from the parental virus in their ability to establish a chronic infection in immunocompetent hosts. Adult mice infected with the wt Armstrong strain cleared the infection within 2 weeks, whereas adult mice infected with the variants harbored virus for several months. These results suggest that the ability of the variants to persist in adult mice is due to enhanced replication in macrophages and/or lymphocytes. This conclusion is further strengthened by the finding that the variants and the parental wt virus grew equally well in mouse fibroblasts and that the observed growth differences were specific for cells of the immune system.     

The role of migration in the evolution of phenotypic switching

Stochastic switching is an example of phenotypic bet hedging, where an individual can switch between different phenotypic states in a fluctuating environment. Although the evolution of stochastic switching has been studied when the environment varies temporally, there has been little theoretical work on the evolution of phenotypic switching under both spatially and temporally fluctuating selection pressures. Here, we explore the interaction of temporal and spatial change in determining the evolutionary dynamics of phenotypic switching. We find that spatial variation in selection is important; when selection pressures are similar across space, migration can decrease the rate of switching, but when selection pressures differ spatially, increasing migration between demes can facilitate the evolution of higher rates of switching. These results may help explain the diverse array of non-genetic contributions to phenotypic variability and phenotypic inheritance observed in both wild and experimental populations.     

Characterization of a Pseudomonas putida rough variant evolved in a mixed-species biofilm with Acinetobacter sp. strain C6

Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria was shown to evolve rapidly by natural selection of better-adapted variants in a mixed-species biofilm consortium (S. K. Hansen, P. B. Rainey, J. A. Haagensen, and S. Molin, Nature 445:533-536, 2007). Adaptation was caused by mutations in a wapH homolog (PP4943) involved in core lipopolysaccharide biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter during the catabolism of benzyl alcohol, the sole carbon source. Examination of biofilm development and the dynamics of the wild-type consortium revealed that the biofilm environment became oxygen limited, possibly with low oxygen concentrations around Acinetobacter microcolonies. In contrast to P. putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response to oxygen starvation. A key factor explaining this conditional, nondispersal phenotype is likely to be the acquired ability of the rough variant to coaggregate specifically with Acinetobacter cells. We further show that the P. putida rough variant displayed enhanced production of a cellulose-like polymer as a consequence of the mutation in wapH. The resulting phenotypic characteristics of the P. putida rough variant explain its enhanced fitness and ability to form tight structural associations with Acinetobacter microcolonies.