Delivery of Antifungal Effectors by the Type VI Secretion System: History Edit
Subjects: Microbiology

1. Introduction

Bacteria and fungi are ubiquitous in nature and co-colonise numerous environmental niches. Focussing on the human host, such cross-kingdom interactions are prevalent within the human microbiota, and are commonly associated with biofilms and medically relevant infections [1]. Such interactions may be chemical, physical or occur through alteration of the shared environmental niche, and, importantly, can be synergistic or antagonistic for the species involved. Regarding synergistic interactions, there are several examples of how bacterial and fungal co-infection potentiates host colonisation and virulence [2,3,4], and mixed species biofilms have been shown to create protective environments [5,6]. Conversely, several Gram-negative pathogens, including Salmonella enterica serovar Typhimurium [7], Pseudomonas aeruginosa [8], and the emerging pathogen Acinetobacter baumannii [9], secrete molecules that can kill fungal cells. Perhaps the best characterised is P. aeruginosa and its interactions with an important fungal pathogen of humans, Candida albicansP. aeruginosa preferentially binds to and forms biofilms on hyphal C. albicans cells and kills the fungus through the action of two virulence factors, secreted phospholipase C and redox-active phenazines [8]. P. aeruginosa also produces a quorum signalling molecule which inhibits the yeast to hyphal switch [10], an important virulence trait in C. albicans.
Whilst antagonistic bacterial-fungal interactions are well recognised, the discovery that a bacterium can directly inject antifungal effectors into fungal cells opens up an exciting new research area underpinning polymicrobial interactions. Recently, we reported that the “antibacterial” Type VI secretion system (T6SS) within the opportunistic pathogen Serratia marcescens, is also a potent antifungal weapon [11]. Two dedicated antifungal effectors, Tfe1 and Tfe2, are translocated into fungal cells by the S. marcescens T6SS, ultimately resulting in fungal death [11]. Here we give an overview of the T6SS and mechanism of effector delivery before focussing on the identification and mode of action of the newly discovered Tfe1 and Tfe2 antifungal effectors. We also present evidence that T6SS-dependent antifungal activity is likely to be a widespread determinant of microbial community composition, before finishing with the key questions and opportunities for future research afforded by this exciting new area of biology.

2. Occurrence of Type VI Secretion Systems

The T6SS is a bacterial nano-weapon that can be used to translocate toxic effector proteins directly into neighbouring cells. It is a versatile system that can be used to deliver antibacterial toxins into rival bacterial cells, representing an important means of inter-bacterial competition, but can also be used to deliver effectors which damage or manipulate host cells, representing direct virulence factors [12]. T6SSs are widely distributed in Gram-negative bacteria. It has been estimated that at least 25% of Gram-negative bacteria contain at least one T6SS, most commonly within the α, β and γ-proteobacteria [13,14]. The majority of such ‘classical’ T6SSs appear to have antibacterial activity, whilst some also, or exclusively, possess anti-host activities. More recently, it has become clear that several other distantly-related families of T6SS also exist, sharing a basic mechanism but with differences in the core machinery. One of these divergent T6SSs occurs in the Bacteroidetes, a Phylum including key members of the gut microbiota, and is used for inter-bacterial competition [15,16]. Bacterial species, and indeed individual strains within a species, vary greatly in their complement of T6SSs, from none up to six different T6SSs, whilst the complement of secreted effector proteins is even more variable [12]. In some cases, the same T6SS can be used for two different functions, such as the Vibrio cholerae T6SS which is used against both competitor bacteria and the host [17]. In other cases, different T6SSs fulfil distinct roles, for example in Burkholderia thailandensis, where T6SS-1 is reported to be exclusively antibacterial, T6SS-5 appears to be exclusively anti-host and T6SS-4 has a distinct role in delivering a manganese-scavenging metallophore protein to the extracellular milieuwhere [18,19]. Regarding potential roles for T6SSs in mediating bacterial-fungal interactions, it is noteworthy that many bacterial species which co-exist with fungi possess T6SSs. Considering bacterial species involved in medically-relevant bacterial-fungal interactions, P. aeruginosaA. baumanniiS. TyphimuriumEscherichia coli and Burkholderia cenocepacia, possess well-characterised T6SSs [20,21,22,23,24]. Similarly, many plant-associated bacteria, including plant growth promoting Rhizobia, biocontrol organisms such as Pseudomonas fluorescens and Pseudomonas putida, and plant pathogens including Agrobacterium tumefaciens and Pectobacterium species, contain T6SSs [25].

3. Effector Delivery by the Type VI Secretion System

The T6SS is a large, multiprotein machinery anchored in the inner and outer bacterial membranes. It uses a contraction-based mechanism to propel an arrow-like puncturing device decorated with multiple effector proteins out of the bacterial cell and into neighbouring target cells. In this way, effectors are delivered inside the targeted cell, from where they exert their toxic activities [12]. The mechanism of this machinery, which is related to that of contractile bacteriophage tails, has been reviewed extensively [26,27,28,29] and the current model will be summarised here (Figure 1). The expelled puncturing device comprises a tube made of stacked rings of the Hcp protein, tipped with a sharp spike made of a trimer of VgrG proteins and one PAAR protein. To achieve “firing” of this structure, a membrane complex is assembled across both bacterial membranes, which then serves as a docking site for the cytoplasmic baseplate complex, within which sits the VgrG-PAAR spike. The Hcp tube can then be assembled onto the spike, extending out across the bacterial cytoplasm. A sheath-like structure made up of the TssBC proteins simultaneously assembles around the Hcp tube in an extended, high-energy conformation, with the two structures linked by a “cap” at the distal end. A rapid and powerful sheath contraction event then drives the Hcp-VgrG-PAAR structure through the baseplate and membrane complex, out of the bacterial cell and into a suitably positioned recipient cell, followed by effector release inside the targeted cell. The contracted sheath is disassembled by a dedicated ATPase, TssH, and further rounds of T6SS assembly and firing can occur.
 
In order to achieve effector delivery, effectors associate with the Hcp-VgrG-PAAR structure in a number of ways [12]. They can interact non-covalently with the inside of the Hcp tube or with the outside of the VgrG-PAAR spike. Alternatively, VgrG, Hcp and PAAR proteins may possess additional effector-containing domains, normally at their C-termini. To accommodate multiple different effector proteins, T6SSs typically contain multiple copies of Hcp, VgrG and/or PAAR proteins and often possess effector-specific chaperones to aid loading of the effector onto the machinery. The T6SS appears to be a very flexible delivery machine, able to deliver effectors of a variety of sizes, structures and functions. These range from antibacterial effectors with cell wall hydrolase, phospholipase and nuclease activities, through metal scavenging proteins, to anti-host effectors with actin modification, inflammasome modulation and membrane fusion functions [12,30,31].

4. Identification of T6SS Antifungal Effectors

The first indications that the T6SS may play a role in bacterial-fungal interactions were reported for plant-associated bacteria, which frequently share their habitat with symbiotic or disease-causing fungi [32,33]. More specifically, the plant-associated Pseudomonad, P. fluorescens Pf29Arp, which has been shown to protect wheat roots from the pathogenic fungus Gaeumannomyces graminis var. tritici, exhibited increased expression of T6SS genes if cultured on fungus-infected roots compared with healthy roots [33,34]. In addition, direct T6SS-dependent activity against both bacterial and fungal competitors was reported for the pathogenic phytobacterium Pseudomonas syringae pv. tomato DC3000 [35]. However, in both cases, no antifungal T6SS effectors were identified. In contrast, effector-based antifungal activity was reported for the T6-secreted protein Tse2 of P. aeruginosa when overexpressed ectopically in the model yeast S. cerevisiae [36,37]. Similarly, transfection of HeLa cells with Tse2 led to cell-rounding. However in physiologically more relevant co-culture experiments, when Tse2 would be delivered by the T6SS, Tse2 toxicity was restricted only to other bacteria [37]. Subsequent structural analysis of Tse2 revealed identity with ADP-ribosyltransferase toxins [38], suggesting general cytotoxic activity towards an evolutionary conserved target rather than being an effector deployed against fungal cells.
Identification of the first fungal-specific T6-secreted effectors was recently reported for the model strain, Serratia marcescens Db10 [11]. S. marcescens possesses a single potent antibacterial T6SS, which has been shown to deliver at least eight distinct antibacterial effectors, and is post-translationally regulated to fire in an offensive manner [39]. Co-culturing S. marcescens with the model yeast S. cerevisiae, or the opportunistic pathogenic fungi C. albicans and C. glabrata, resulted in T6SS-dependent inhibition of fungal growth. This effect was dependent on cell-to-cell contact, dispelling doubts about whether the T6SS could breach the thick and rigid fungal cell wall.
To identify the effectors responsible for antifungal activity, initial experiments focussed on T6-secreted proteins which had previously been identified in a proteomics screen comparing the secretome of wild type S. marcescenswith that of a T6SS-inactive mutant [40]. Of these, only one, Ssp3, exhibited fungicidal activity against C. albicans. Interestingly, Ssp3 was initially classified as an antibacterial toxin, since overexpression in E. coli produced modest toxicity, which was alleviated upon co-expression of a small open reading frame situated directly upstream of the toxin [40]. However, deletion of this immunity gene, sip3, in S. marcescens did not affect viability, and no inhibition of the Δsip3 mutant upon co-culture with strains able to perform T6SS-mediated delivery of Ssp3 was observed, indicating that Ssp3 is not an antibacterial effector under such conditions [11]. This is in contrast with true T6SS antibacterial effectors, where mutants lacking the cognate immunity protein are non-viable due to self-killing and are susceptible to delivery of the cognate effector upon co-culture with a wild type strain. To reflect its antifungal rather than antibacterial activity and its identification as the first T6SS-secreted antifungal effector protein, Ssp3 was renamed Tfe1. However, deletion of tfe1 in S. marcescens did not reduce antifungal activity against S. cerevisiae or C. glabrata in co-culture experiments, suggesting the existence of additional antifungal effectors. To identify such effectors, a second proteomics approach was employed to capture the cellular proteome, rather than the secretome, with the rationale that a T6SS-inactive mutant would retain potential effector proteins inside the cell. Analysis of the cellular proteome of a ΔtssE mutant compared with wild type S. marcescens revealed twelve proteins that displayed increased abundance in the T6SS-inactive mutant. Nine were proteins already known to be secreted by the T6SS, including components of the puncturing structure and Tfe1, whilst the remaining three proteins were unlikely to be antibacterial effectors since they were not encoded next to potential cognate immunity proteins. Subsequently, mutational analysis identified one of these three candidates as the second antifungal effector, Tfe2 [11]. Notably, whilst Tfe2 failed to exhibit antibacterial activity, loss of Tfe2 virtually abolished S. marcescens antifungal activity against S. cerevisiae and C. glabrata. Thus the “antibacterial” S. marcescens T6SS is also a potent antifungal weapon, able to kill S. cerevisiae and Candida spp. by delivering two dedicated antifungal effectors Tfe1 and Tfe2.
Given rapid recent advances in the T6SS field, relating to both its structure and secretion mechanism [41,42,43,44] and the diversity of secreted proteins [12], it is perhaps surprising that antifungal-specific effectors have been identified only recently [11]. This is likely due to the fact that the strategies used to discover novel effectors have been based on the assumption that T6SSs are primarily employed as weapons in interbacterial warfare or else as classical virulence factors against higher eukaryotes. Therefore, effector identification strategies have often relied on their presumed antibacterial activity and the presence of cognate immunity proteins. For example, using unbiased proteomics approaches, only those T6-secreted proteins harbouring a potential immunity protein were considered candidate T6SS effector proteins, with confirmation being based on their ability to kill a non-immune sibling strain [37,40]. Similarly, effector identification via random transposon mutagenesis and deep sequencing (Tn-seq) in wild type and T6SS inactive mutant backgrounds relied on the identification of immunity proteins as being essential in the presence of a functional T6SS [45]. However, by using such strategies antifungal effectors would be missed. Indeed, no immunity protein is associated with Tfe2, consistent with the premise that bacteria deploying fungal-specific toxins would not require cognate immunity proteins for protection. Furthermore, hypothesis-driven in silico discovery of T6SS effectors, based on domain and homology screens (e.g., peptidoglycan-degrading enzymes [46], lipases [47] and MIX-motif containing proteins [48]), are led by the prior knowledge of previously identified effectors and thus are likely to miss novel fungal-specific effector proteins. Alternatively, effectors are often located within T6SS gene clusters or distant loci encoding additional Hcp, VgrG and/or PAAR proteins, and so candidate effectors can be identified by their genetic context. Similarly, candidate effector domains fused with VgrG or PAAR proteins are readily identifiable through bioinformatic analysis. However, these approaches will miss effectors located in genomic regions otherwise unrelated to T6SS, such as Tfe1 and Tfe2, and to-date have again typically relied on identification of immunity proteins and/or demonstration of antibacterial activity as validation as bona fide T6SS substrates.
It is also important to note that antifungal effectors (like antibacterial or anti-host effectors) may be delivered by T6SSs that are silent under laboratory conditions, thus hindering their identification. Knowledge about signalling pathways and regulatory mechanisms, in combination with genetic tools to generate constitutively active T6SSs, may prove instrumental in the discovery of future T6SS effectors. This has been exemplified by deletion of the gene encoding for the sensor kinase RetS in P. aeruginosa or the quorum-sensing master regulator OpaR in V. parahaemolyticus, both rendering the respective T6SSs constitutively active [49,50]. Similarly, interfering with the phosphorylation status of the scaffolding protein Fha1 by deleting the Ser-Thr phosphatase PppA in P. aeruginosalocks the T6SS in its active state, which allowed for identification of T6-secreted proteins under standard laboratory conditions [51]. Future strategies to study such silent T6SSs will likely require a combination of genetics tools together with performing co-culture experiments under physiologically relevant conditions. In this regard it is notable that the laboratory silent T6SSs of V. cholerae O1 serogroup strains C6706 and N16961 are stimulated by intestinal factors, namely, mucins and bile salts [52].
 

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