The trans-kingdom identification of negative regulators of pathogen hypervirulence

Abstract

Modern society and global ecosystems are increasingly under threat from pathogens, which cause a plethora of human, animal, invertebrate and plant diseases. Of increasing concern is the trans-kingdom tendency for increased pathogen virulence that is beginning to emerge in natural, clinical and agricultural settings. The study of pathogenicity has revealed multiple examples of convergently evolved virulence mechanisms. Originally described as rare, but increasingly common, are interactions where a single gene deletion in a pathogenic species causes hypervirulence. This review utilised the pathogen-host interaction database (www.PHI-base.org) to identify 112 hypervirulent mutations from 37 pathogen species, and subsequently interrogates the trans-kingdom, conserved, molecular, biochemical and cellular themes that cause hypervirulence. This study investigates 22 animal and 15 plant pathogens including 17 bacterial and 17 fungal species. Finally, the evolutionary significance and trans-kingdom requirement for negative regulators of hypervirulence and the implication of pathogen hypervirulence and emerging infectious diseases on society are discussed.

Keywords: antimicrobial resistance; bacteria; emerging infectious diseases; fungi; pathogen; virulence.

Figure 1.

The different ways in which hypervirulence and increased pathogen dissemination can contribute to disease outbreaks. Presented is the virulence phenotype of wild-type and mutant pathogens within the host organism and subsequently in the environment post dissemination (adapted from Ten Bokum et al.2008). Hypervirulent mutants multiply at the same rate, or faster, within the host, reaching a higher final pathogen burden. Mutants with an increased capacity to be disseminated beyond the host can also cause a rise in pathogen burden, transmission and disease incidence, even when in a wild-type pathogenic background. The combination of hypervirulence and increased dissemination poses the greatest threat to the host organism. In contrast, mutants with reduced dissemination capacity, reduced persistence or attenuated virulence will result in a decline in pathogen burden.

Figure 2.

The trans-kingdom distribution of 112 negative regulators of pathogen hypervirulence. (A) The phylogenetic distribution of the negative regulators of hypervirulence, identified within the pathogen–host interactions database (PHI-base), including bacterial, protozoan, fungal and nematode pathogens. The tree was generated using the NCBI taxonomy tree building service (Sayers et al.2009). (B) The number of hypervirulent mutations per pathogenic species subdivided into animal (n = 67) and plant (n = 45) attacking pathogens.

Figure 3.

The top 20 biological, molecular and cellular functions of the 112 negative regulators of pathogen hypervirulence. Gene ontologies were identified using the Blast2GO software (Gotz et al.2008).

Figure 4.

Masking pathogenic potential. Presented are two examples of how the composition of the exterior surface of a bacterial and a fungal pathogen is essential to macrophage colonisation. (A) The M. tuberculosis infection cycle within a mammalian host, including the establishment of long-term persistent infections via macrophage colonisation. (B) The role of the M. tuberculosis cell wall in the modulation of host immune responses. Hypervirulence can occur as a consequence of either an elevated or impeded immune response caused by alterations to cell wall composition. (C) The fungal polysaccharide capsule and the melanised cell wall of C. neoformans promote mammalian infection and macrophage colonisation. (D) The absence of negative regulators of the MAPK and cAMP-dependent PKA pathways in C. neoformans, which control capsule and melanin biosynthesis, causes hypervirulence. Purple denotes proteins whose absence results in a hypervirulence entry into PHI-base. Abbreviations: PM = Plasma membrane, GPCR = G-protein coupled receptor, Cac1 = Adenylate cyclase, Pka1/2 = PKA catalytic subunits, Pkr1 = PKA regulatory subunit, MAPK = Mitogen-activated protein kinase, Ste11 = MAPKKK, Cpk1 = MAPK, Crg1 = Regulator of G-protein signalling.

Figure 5.

Intercellular communications promote strength in numbers. Presented are two examples of how bacterial and fungal cell-to-cell communication influences community structure and virulence. (A) The development of the P. aeruginosa bacterial biofilm and the impact of biofilm dispersal in mammalian hosts during the chronic and acute phases of infection. (B) Quorum sensing (QS) in P. aeruginosa regulates biofilm dispersal and toxin biosynthesis, while the absence of various negative regulators of QS results in hypervirulence. (C) QS in Candida species regulate fungal morphology in addition to biofilm formation and maturation. (D) The absence of two transcription factors, Efg1 or Ace2, regulated by cAMP-dependent PKA and RAM signalling pathways, controls biofilm formation and causes hypervirulence. Purple denotes proteins whose absence results in a hypervirulence entry into PHI-base. Green represents proteins whose absence results in an increase in chronic infection and not hypervirulence. Panel A abbreviations: Pel/Psl = Extracellular polysaccharides, T3SS = Type III secretion system, T6SS = Type VI secretion system. Panel B abbreviations: CW = Cell wall, PM = Plasma membrane, LasR/RhlR = LuxR-type QS receptors, LasI/RhlI = LuxI-type QS synthases, AHL = Acylhomoserine lactone (purple circle), BHL = N-butyrylhomoserine lactone (blue square), RsaL/QslA/QscR = QS repressors. Panel D abbreviations: GacS/GacA = Two component system sensor and regulator, RetS = Orphan sensor and repressor of GacS, RsmA = Transcriptional repressor. Panel D abbreviations: GPCR = G-protein coupled receptor, Cyr1 = Adenylate cyclase, PkA = PKA complex, Efg1/Ace2 = Growth regulating transcription factors, RAM = Regulation of Ace2 and morphogenesis pathway.

Figure 6.

The pathogenic balance: morphogenesis and growth rate. Presented are two examples of how a bacterial and a fungal pathogen restrict pathogenic morphogenesis and growth to maintain a highly adapted intracellular or a tightly coordinated intercellular form of infection. (A) Salmonella enterica serovar Tryphimurium pathogenicity islands promote mammalian host invasion, intracellular macrophage colonisation and systemic infection. (B) SciS limits intracellular growth within macrophages during late infection and the absence of SciS causes increased intracellular replication and hypervirulence. (C) In the fungus, E. festucae, the NOX complex and related signalling pathways coordinate mutualistic grass colonisation and prevent pathogenicity. (D) The absence of a single NOX component, excluding Cdc24, causes increased ROS production, pathogenic hyphal growth, and results in hypervirulence. Purple denotes proteins whose absence results in a hypervirulence entry into PHI-base. Panels A and B abbreviations: SPI1 and SPI2 = Samonella pathogenicity island 1 and 2, SsrA = TCS sensor, SsrB = TCS regulator, SciS = Salmonella centisome 7 island. Panel D abbreviations: BemA = Scaffold protein involved in cell polarity and orthologue of Saccharomyces cerevisiae Bem1, NoxA = NADPH oxidase, NoxR = p67phox-like regulator, RacA = Small GTPases, Cdc24 = Guanine nucleotide exchange factor, SakA = Stress-activated MAPK, SOD = Superoxide dismutase, ROS = Reactive oxygen species.

Figure 7.

Negative regulators of toxin biosynthesis. Presented are two examples of how alterations to the regulation and biosynthesis of secreted virulence factors can give rise to hypervirulence in a bacterial and a fungal pathogen. (A) The contrasting profiles of hospital (HA)- and community (CA)-acquired methicillin resistant Staphylococcus aureus (MRSA). (B) A single gene on a mobile genetic element accounts for differing virulence profile of HA-MRSA and CA-MRSA. The psm-mec gene encoded on the SCCmec mobile genetic element suppresses AgrA-mediated quorum sensing and attenuates virulence in HA-MRSA, while the absence of psm-mec in CA-MRSA results in hypervirulence. (C) Fusarium graminearum secreted a trichothecene mycotoxin, DON to inhibit host plant defence responses promoting infection. (D) The natural emergence of a hypervirulent 3A-DON chemotype with increased fitness. In addition, the absence of a cytochrome P450 monooxygenase or a partial terpene cyclase results in the transcriptional derepression of DON mycotoxin biosynthesis, causing increased DON secretion and hypervirulence. Purple denotes proteins whose absence results in a hypervirulence entry into PHI-base. Panel A abbreviation: PIA = Polysaccharide intercellular adhesin. Panel B abbreviations: QS = Quorum sensing, ArgA = Response regulator of the Arg two component system. Panel D abbreviations: TRI = Gene encoding protein involved in trichothecene biosynthesis, Tri15 = Transcriptional repressor of TRI gene cluster.

Figure 8.

Anthropogenic and environmental factors driving the evolution of emerging infectious diseases. Specific anthropogenic and environmental factors are increasingly having an impact on interactions between pathogen and host, driving rapid evolutionary changes within the pathogen that can give rise to a hypervirulence phenotype, new diseases, altered host range and/or antimicrobial resistance. Subsequently, the same anthropogenic and environmental factors can facilitate the dissemination and rise in prevalence of the newly adapted pathogens, representing the increasing risk of EIDs, threatening human and animal health, agriculture and natural ecosystems.