Perlin Lab Research

EVOLUTION OF HOST/PATHOGEN INTERACTIONS: MECHANISTIC INSIGHTS

The research in my lab focuses on the evolution of interactions between pathogens and the hosts on which they cause disease. At present, this work has two main areas of emphasis: fungal/plant interactions and population dynamics of bacteria resistant to antibiotics.

 

A. Fungal/Plant Interactions leading to Pathogenesis

Our work with fungal/plant interactions has examined primarily the “smut” fungi.

Plant Diseases Caused by Smut Fungi:

M. violaceum

Microbotryum violaceum

"anther smut"

and

Infection of Maize by U. maydis

Ustilago maydis

"corn smut"

These are basidiomycetous fungi that have several different morphological stages in the course of their development. The final stage is obligately parasitic, in that production of the diploid spores requires successful infection of a suitable host plant. The general lifecycle is illustrated below.

The Smut Lifecycle

Questions of Host Specificity and Evolution of Sexual Systems

We began our work with smut fungi over 20 years ago with the anther smut, Microbotryum violaceum, a fungus that infects members of the carnation family (Caryophyllaceae) and replaces the pollen of anthers with its own teliospores. Our early work attempted to establish such molecular methods as genetic transformation and provided DNA “fingerprints” for individual isolates from different host species. Since then it has become clear that what has been called Microbotryum violaceum (or Ustilago violacea, Pers. Roussel ) for hundreds of years is actually a species complex, comprised of many different species or incipient species, each of which is adapted to infection of its own host species. What has emerged is an exciting system for examination of the ecology and evolution of host/pathogen interactions in “wild” non-agricultural environments, a model for studying the evolution of sexual systems and sex chromosomes, and gene expression during infection that may give clues to important genes involved in pathogenicity. http://www.amherst.edu/~mhood/mv/community.htm

 

Fungal Dimorphism and the Link to Pathogenicity

A major aspect of our work with fungi involves genes that control morphogenesis and pathogenicity in fungi. One link that ties these two processes together is the dimorphic switch between yeast-like and filamentous forms. In model organisms such as the ascomycete yeast, Saccharomyces cerevisiae, and the basidiomycete smut, Ustilago maydis, membrane proteins that sense and transport ammonium have been implicated in this switch. The MEP proteins from yeast have been intensively studied, while those in other fungi, particularly pathogenic organisms, have been much less scrutinized.

 

The yeast S. cerevisiae can adopt several alternative developmental fates depending on the availability of specific nutrient sources. When nitrogen and fermentable carbon sources are both plentiful, haploid and diploid yeast cells reproduce by budding. When nitrogen is limiting but an abundant fermentable carbon source is present, diploid yeast cells undergo pseudohyphal differentiation (i.e., a dimorphic switch) to form filamentous colonies that forage for nutrients. These alternative fates allow this nonmotile organism to appropriately adapt to its surroundings. The signaling cascades by which yeast cells sense and respond to nutrients serve as models to understand how all cells sense and respond to the environment. Ammonium is a preferred nitrogen source for many fungal and bacterial species and for plants, as well. Moreover, the ability of microbes to sense and transport nitrogen is critical, not only for survival, but also as a prelude to a variety of developmental processes. These responses to nutrient availability are governed by both a mitogen-activated protein kinase (MAPK) cascade and the cAMP-dependent protein kinase pathway, with cross-talk between these parallel pathways at several important points; these cascades also control several aspects of pathogenic development discussed below. How nutrient signals in the extracellular environment control the activation of these globally conserved signaling cascades is a central question.

 

1. Nutrient sensing controls pathogenicity

Many fungal pathogens utilize a similar switch between a yeast-like form and a filamentous form as an integral part of their overall strategy of disease production. For example, the dimorphic transition from budding yeast to filamentous forms is crucial for infection for Candida albicans, and it is important for Cryptococcus neoformans. Similarly, in some plant pathogens, such as the maize pathogen Ustilago maydis, the dimorphic switch plays a critical role in both morphogenesis and pathogenicity. In fact, there is a high degree of conservation in the signaling cascades controlling development and virulence of these divergent ascomycete (S. cerevisiae, C. albicans) and basidiomycete (C. neoformans, U. maydis) yeasts. Based on these conserved pathways, studies in the model yeast S. cerevisiae have provided insight into fungal pathogenesis in humans and plants. In turn, studies in the pathogens will not only validate studies from yeast but also are expected to begin to identify universal and species specific components of ammonium sensing networks.

 

Different filamentous forms of U. maydis

Several factors can cause cell differentiation in Dimorphic Fungi

  • Mating Pheromones
  • Nutrients (low N)
  • Host Cues
Nutrient sensing in fungal plant pathogens

As mentioned above, many of the conserved features of signaling are also found in fungal plant pathogens. The smut fungus U. maydis exists as haploid yeast-like sporidia, and mating of cells of opposite mating-type, accompanied by signals from host plants, leads to formation of infectious dikaryotic hyphae. U. maydis infection causes tumor formation on all maize shoot tissues, including tumor formation, with particular severity on the ears. Such damage can result in annual losses exceeding $200 million. For U. maydis mating occurs readily on rich media, but haploid or diploid sporidia grow filamentously on nitrogen starvation medium. The first genes encoding proteins involved in ammonium transport were cloned from yeast and Arabidopsis. As mentioned above, for S. cerevisiae, low nitrogen in the presence of an abundant fermentable carbon source leads diploid yeast cells to grow as pseudohyphae. The genes primarily responsible for both transport and sensing of available nitrogen include MEP1, MEP2, and MEP3, each of which encodes an integral membrane protein that transports ammonium ions or the toxic analog methylammonium. Of the three,Mep2 is a high-affinity permease, that acts as both an ammonium transporter and sensor. The yeast Mep2 protein controls the transition to pseudohyphal growth in response to ammonium limitation. mep2/mep2 mutant strains have no vegetative growth defect on nitrogen limiting medium, but in contrast to wild type cells are unable to undergo pseudohyphal differentiation. This defect can be suppressed by exogenous cAMP or by dominant activation of the PKA signaling pathway, supporting models in which Mep2 either functions upstream of PKA signaling, or the two pathways function in parallel. In U. maydis, we have identified two homologs of Mep2, called Ump1 and Ump2, and importantly Ump2 is required for filamentous growth on low nitrogen medium and can restore both growth and filamentous growth when heterologously expressed in S. cerevisiae mep1,2,3 mutant strains. Ump2 was identified as being more highly expressed under conditions of low ammonium. Thus, the S. cerevisiae Mep and U. maydis Ump proteins are physical and functional homologs of each other and function in both ammonium transport and ammonium sensing. The yeast Mep and fungal Ump proteins are part of a larger family of proteins that are conserved between bacteria, yeast, fungi, and humans.

2. Regulation of transporter function by phosphorylation

Several examples exist where proteins involved in transport are regulated by phosphorylation/dephosphorylation cycles. Our preliminary work examining the phosphorylation of the yeast Mep proteins indicates that a putative PKA site is present. Furthermore, the target residue is necessary for appropriate Mep function to control filamentation in both S. cerevisiae and U. maydis. Our ongoing studies aim to define the function of this phosphorylation, and will provide insight into conserved regulatory mechanisms that operate in these and other sensory receptors. We are also using split-ubiquitin and protein association assays to identify interactions between the Ump proteins and possible signaling components of U. maydis.

3. Additional Roles for Signaling Pathway Components

Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3epsilon and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis, as follows. Rac1 sequesters Cla4 to the growing tip. Thus, Rho1 could interfere with Rac1 activities by preventing Rac1 from localizing at the growing pole. Other possibilities for how Rho1 could indirectly act as negative regulator of Rac1 are by sequestering Cdc24 (the RhoGEF) or Cla4 away from Rac1. Yeast two-hybrid analyses suggest possible interaction between Cdc24 and Rho1. As with Rho1, Cdc24-null cells are nonviable. In vivo, Rho1 and Rac1 could compete for Cdc24. On the other hand, Cla4 has been shown to localize at the bud-neck in S. cerevisiae. According to our GFP-Rho1 data, Rho1 also localizes (possibly with the help of Pdc1) at the bud-neck or in the septation area. Thus, it is a possibility that Rho1 is somehow involved with sequestration of Cla4 to the septation area and away from the growing tip and Rac1. In such cases, cells would then undergo budding instead of filamentation.

 

 

 

B. Evolution of Antibiotic Resistance in Bacterial Populations

Microbial diversity is important in a variety of contexts, in particular having implications for infectious diseases, bioremediation, and environmental engineering. Examination of the mechanisms underlying such diversity and its evolution are important for providing a roadmap that can lead to better understanding of the above-mentioned and other, related, areas. Several examples are available where mutualism in microbial communities can lead to the maintenance of microbial diversity. Fewer examples have been presented where some individuals provide protection to others in a population without a concomitant benefit being returned to the protector. Protection of sensitive members of a population against antimicrobials by resistant genotypes has been observed in biofilms. In these cases, spatial proximity to the protector/producer was a prerequisite, or at least an important component, for survival by otherwise sensitive individuals. We have extended these studies to shaking liquid or planktonic populations in order to examine the dynamics of such unrewarded protection or altruism. We have developed a family of mathematical models that have examined antibiotic resistance in such systems (Dugatkin et al., 2003, 2005) and have generated preliminary data which support some aspects of these models (Dugatkin et al., 2004). Our results so far show that such altruists can provide frequency-dependent antibiotic resistance to other members in the population. Furthermore, frequency-dependent selection of the traits of these altruists promotes microbial diversity. We are currently conducting more detailed experiments designed to test our model's predictions, and will then use the results obtained to further refine the models. Specifically, we will use competitions between near-isogenic Escherichia coli strains that are either sensitive to ampicillin or are resistant, by virtue of a plasmid-encoded b-lactamase. Among those strains that are resistant, we compare the outcome for those strains that only protect themselves from ampicillin versus those strains that can also provide protection to others in their vicinity, i.e., by destroying ampicillin nearby in the medium. These analyses have also been extended to competition experiments with greater relevance for natural clinical or human settings. Specifically, we have examined whether ampicillin-resistant E. coli can protect sensitive Salmonella in their vicinity. Our results (Perlin et al., 2009) demonstrate clearly that non-pathogenic antibiotic-resistant bacteria may protect otherwise susceptible pathogens by a mechanism that does not involve gene transfer. Evolution of such transient survival mechanisms may thereby hinder therapeutic use of antibiotics, and should be considered in devising effective treatment strategies.

Altruists

CURRENT LAB MEMBERS

 

Su San Toh, graduate student. Examining gene expression in plants infected with M. violaceum.

Michael Cooper, graduate student. Exploring nitrogen metabolism in U. maydis.

R. Margaret Wallen, graduate student. Examining the interaction between ammonium transport and mating in U. maydis.

Seth Adams, graduate student. Characterizing mutations that affect appressorium development and possible impacts on pathogenicity in U. maydis.

Swathi Kuppireddy, graduate student. Development of gene deletion technology for M. violaceum and examining the role of genes in this organism via such deletions.

Hannah Mianzo, graduate student. Exploring relationships between signaling components and the b mating locus in U. maydis.

Ming-Chang "Nelson" Tsai, graduate student. Studying roles of gene expression for various target genes in M. violaceum.

Jared Andrews, undergraduate, Honors student. Characterizing the roles of sugar transporters in cell morphology and mating in M. violaceum.

Dominique Razeeq, undergraduate. Characterizing the roles of secretory lipases in cell morphology and mating in M. violaceum.

Trisha H. Patel, undergraduate. Member of the team identifying interacting proteins with signaling components from U. maydis that may affect nitrogen metabolism.

Ann-Claude Rakotoniaina, undergraduate. Member of the team examining gene expression of M. violaceum during plant infection.

 

RECENT FORMER STUDENTS OR SOON-TO-BE GRADUATES

 

Ben Lovely, PhD. Examined signaling via Hsl proteins in U. maydis and their connections to other signaling pathways.

Charu Agarwal, PhD. Co-mentored with Dr. David Schultz, Department of Biology. Charu analyzed cAMP turnover in U. maydis via a genetic and biochemical analysis of cyclic phosphodiesterases of the fungus.

Jinny Paul, PhD. Characterization of ammonium transporter homologues of several fungi, with emphasis on those from U. maydis and M. violaceum.

Cau Pham, PhD. Exploring the roles of 14-3-3 and Rho proteins in cytokinesis, cell polarity, morphology, and pathogenesis for U. maydis. Currently a Research Associate at the Centers for Disease Control.

Zhanyang Yu, PhD. Isolation and characterization of Rho1 homologue in U. maydis and identification of interacting proteins and pathways.

Tia Alton, undergraduate, Honors Student. Member of the team working on competition experiments with bacteria. Currently in medical school.

Evan Raff, undergraduate, Honors Student. Member of the team identifying interacting proteins with signaling components from U. maydis. Currently in medical school.

Alexander Bajorek, undergraduate, Honors Student. Member of the team working on competition experiments with bacteria. Currently in medical school.

Courtney McKenzie, undergraduate, Honors Student. Member of the team working on competition experiments with bacteria. Currently in medical school.

Cayse Powell, undergraduate, Honors Student. Member of the team working on competition experiments with bacteria. Currently in medical school.

Himati Patel, undergraduate, Honors Student. Member of the team working on competition experiments with bacteria. Currently in pharmacy school.

Lacey Hazel, undergraduate. Work on developing a gene disruption system for M. violaceum. Currently applying to graduate programs in neurobiology.

Greg Shaw, undergraduate, Honors Student. Member of the team identifying interacting proteins with signaling components from U. maydis. Currently in medical school.

Trisha Patel, undergraduate, Honors Student. Member of the team identifying interacting proteins with signaling components from U. maydis. Currently in medical school.

Anna Hellman, undergraduate, Honors Student. Member of the team identifying interacting proteins with signaling components from U. maydis. Currently in medical school.