Antieukaryotic Type Six Secretion System Virulence Factors of Bacteria

The type 6 protein secretion system (T6SS) is prevalently utilized by Gram-negative bacteria to compete for resources and space. Upon activation, toxic effectors from this secretion system are translocated into the competitor prokaryote or eukaryote in a contact-dependent manner. While much has been reported on T6SS-mediated prokaryotic competition, very little is understood about the mechanisms of bacterial interactions with eukaryotic hosts. Likewise, many virulent T6SS effectors are known to be antibacterial. In recent years, however, evidence has emerged on numerous T6SS effectors that interact with related immunity proteins in a range of eukaryotic hosts. Insights into how this effector-immunity pairing alters the physiological responses of the recipient organism might provide opportunities relating to the T6SS agricultural and biotherapeutic applications. We, therefore, summarize the impacts of the T6SS effectors with a special focus on bacterial interactions with animals, plants, and fungi. We further briefly discuss pipelines that are currently used to characterize antieukaryotic T6SS effectors.


Introduction
Strategies that maximize fitness, survival, and thrive in diverse environments entail numerous common themes in bacteria (e.g., rapid growth and biofilm formation), one of the most important being the type six secretion system (T6SS). This multi-protein secretion system is encoded by large and conserved gene clusters in both pathogenic and non-pathogenic Gram-negative bacteria (GNB) 1 , and controls bacterial interaction with other microbes, plants, and animals. The T6SS is highly diverse in animal-and plantassociated bacteria, including the GNB of the phylum Proteobacteria. As a result, phylogenetic studies have sub-classified the system into the main Proteobacterial T6SS I , Francisella T6SS II , and Bacteroidetes T6SS III cluster loci. 2 The T6SS is analogous to tails of Myoviridae T4 bacteriophages and R-type pyocins, which might have been caused by an endless evolutionary arms-race that governs phage-bacteria interactions, paving way for GNB to repurpose phage gene products integrated into their genomes during infection  Virulence effectors of the T6SS are surprisingly dependent on "themselves" for secretion, lack N-terminal hydrophobic sequence, and sometimes require contact for release. 3 The T6SS and associated effectors shape how bacteria respond to other microorganisms, the host within which they reside, and the environment (Fig 2). This effectively makes the system one of the core regulatory components of interspecies and interkingdom competition in bacteria. However, an in-depth understanding of T6SS-mediated microbemicrobe and host-microbe interactions will come from completing the T6SS structure, which is incomplete due to a lack of high-resolution structural information. 4 As a result, several projects are underway to report on the structures that may provide important steps toward a complete atomic model of T6SS assembly. 5-7 Several reports have established that the T6SS delivers a diverse group of antibacterial and antieukaryotic effectors (Table 1), which promote interspecies and interkingdom communication. Such a communication network appears to be essential for bacteria to respond to fluctuations within dynamic environmental conditions. Beyond this, bacteria, being social organisms, can use T6SS to constantly make physical contact with various eukaryotic hosts over space and time, resulting in either a mutualistic or parasitic lifestyle.
A range of conditions can determine whether the former or the latter lifestyle prevails over the other. But with increasing discoveries relating to the participation of secretion system effectors, the molecular exchange of proteins during bacterial-eukaryotic interactions (BEIs) will serve as one of the pivotal determinants of antieukaryotic or symbiotic relationships. The crucial role of molecular mechanisms governing BEIs will likely extend beyond human to plant infecting fungal pathogens in as far as secretion systems are used in defining these interactions. 8 Our understanding of how the transfer of certain compounds such as nutritive 3 metabolites, proteins and nucleic acids, which enable cell-cell communication, facilitate 4 BEIs may be far ahead our understanding of how secretion systems and bacterially 5 secreted effectors contribute to this intricate dialogue. Nonetheless, evidence in support 6 of the role of T6SS in BEIs is still not as forthcoming as for other systems despite the 7 widespread occurrence of T6SS in GNB and the fact that bacteria live in close 8 associations with eukaryotes. Given the anticipated wealth of information on the 9 ecological importance of T6SS, we provide the current state of the knowledge on how this 10 system influences the various collaborative scenarios involving bacteria with animals, 11 plants, and fungi. 12 13

Current understanding of T6SS effectors and functional diversity 14
The functional spectrum of a secretion system is defined by the proteins it secretes and 15 the outcomes thereafter. 52 Different T6SS gene clusters are present in different GNB or 16 in the same bacterium to encode T6SS, that is assembled into a metaphorical "crossbow" 17 consisting of bacterially conserved 13 core structural components. 53 These components 18 include TssA-TssM and Proline-Alanine-Alanine-Arginine (PAAR) motif-containing 19 proteins that form the membrane-bound sub-assemblies of one or several complex 20 injection systems. Most of the bacteria encoding the T6SS clusters are regarded as 21 prolific gene exchangers. 54 In the case of single-copy clusters, the two major T6SS 22 effector genes, hemolysin co-regulated protein (Hcp), a ring-shaped hexamer that 23 recognizes cognate effectors, and valine glycine repeat G (VgrG), used to puncture 24 neighboring cells, , are commonly found as duplicate or sometimes as more copies in the 25 genome of GNB. 11 Clusters that are found within the same organism are not capable of 26 completely substituting one another, although effector redundancy has recently been 27 reported. 56 Therefore, a lack of cluster redundancy may suggest that one organism must 28 employ different T6SS clusters to attack different targets. This way, bacterial interactions 29 mediated by the T6SS can be cooperative, specialized, or competitive. Virulent T6SS 30 effectors can kill both prokaryotic and eukaryotic cells. To this effect, deadly combinations 31 of specific effectors can be loaded at different positions directly or indirectly via adapters 32 to Hcp, docked to VgrG via the Proline-Alanine-Alanine-Arginine or PAAR motif-33 containing module, or as functional C-terminals of evolved Hcp, VgrG or PAAR proteins. 4 34 Additional consequences of functional diversity include opposing views about whether 35 T6SS effectors are that different from toxins secreted by this system. Effector proteins of 36 GNB are defined by their dependency on a secretion system for delivery into eukaryotic 37 targets and exertion of a "subtle" effect on the target cell. In some instances, this occurs 38 within a network of effectors delivered by the same system to suppress or manipulate the 39 target in a certain way beneficial to the pathogen for a time. oxygen species generation in target cells in a manner similar to when bacteria are 44 exposed to antibiotics. 59 In respect to this mechanism, bacterial T6SS effectors may be 45 associated with "brute force" or lethality, causing irreversible effects and exhibiting high 46 specificity in their biochemical role. Surprisingly, contrary to effector secretion system 47 dependency, the effects of a bacterial toxin can be observed on living cells through 48 exogenous addition. 60 Based on conflicting descriptions and mechanisms, the T6SSs, 49 therefore, appear to secrete toxin-like effector proteins with a variety of cellular activities, 50 including degradative enzymes, transcription factors, and hormones. This suggests the 51 use of both brute force and stealth force by GNB based on arising needs. 61 52 53

Antianimal T6SS effectors 54
The T6SS is generally perceived to be a dedicated antibacterial weapon (Table 1) 55 (Reviewed in 62 ). However, in addition to antibacterial effectors, discoveries of 56 antieukaryotic, and those that have a dual role called trans-kingdom effectors, have been 57 made. 63 The latter is evidenced by effectors, such as Secreted small protein (Ssp3/Tfe1), 58 which can target both prokaryotic and eukaryotic cells (Table 1). Trans-kingdom effectors 59 possess phospholipase activity and oftentimes target the cell membrane. These 60 membrane targeting phospholipases form pores that affect cellular integrity (Table1) and 61 include Type VI secretion exported effector L (TseL)/Type six lipase effector 2 (Tle2), 62 Type VI secretion exported effector 5 (Tse5), and Virulence-associated secretion protein 63 (VasX) ( Table 1). It is possible that the ability of these effectors to act in trans is due to cholerae carries a C-terminal effector domain that cross-links actin, impairing the 94 phagocytic activity of host cells. 67 Taken together, these studies suggest that the T6SS 95 has somehow evolved for competition with diverse eukaryotic hosts (Fig 2). also has been reported that the expression of the T6SS is among several regulatory 106 mechanisms regulated by environmental cues that mimic host conditions. 68,71 Most 107 T6SSs cannot be used to target Gram-positive bacteria (GPB) because their 108 peptidoglycan is too thick to puncture. However, it is likely that more GPB adapted 109 effectors are yet to be discovered. 72  operating under these regulators (e.g., Serratia phospholipase PhlA and the pore-forming 150 toxin ShlA) are not involved in the actual killing of the fungi. 77 In addition to that, secreted 151 S. marcescens chitinases, which are highly produced by this bacterium, were found not 152 to be involved in the killing of the mycelia. Taken together, these results point to other 153 mechanisms that could be involved in the killing of fungal cells during the interaction 154 between S. marcescens and fungi (Fig 2). 155 Of recent, the T6SS of S. marcescens was strongly implicated in killing the cells of the 156 ubiquitous yeast Saccharomyces cerevisiae, and human fungal pathogens belonging to 157 the genus Candida (i.e., C. albicans and C. glabrata). 43 This may partly explain the killing 158 of fungi of the phylum Zygomycota by S. marcescens as previously discussed. 77 Indeed, 159 the strongest indications that the T6SS can manipulate BFIs comes from the fact that S. 160 marcescens directly translocate two T6SS fungicidal effectors, Tfe1 and Tfe2 (Tfe for 161 T6SS antifungal effector) ( Table 1). Initial evidence relating to functioning of these 162 antifungal effectors suggests that Tfe1 impairs nutrient uptake and amino acid 163 metabolism and induces autophagy, while Tfe2 impairs membrane potential but without 164 introducing any pores in this organelle. 29 Further analysis of these T6SS antifungal 165 effectors will likely provide key insights regarding BFIs in the context of polymicrobial 166 communities. With the help of tools that are currently being developed, including 167 bioinformatics approaches as discussed below, we are likely to witness identification of 168 more T6SS antifungal effectors in future, not just from S. marcescens but other bacteria. 169 Pipelines currently assisting the discovery of antieukaryotic T6SS effectors 178 While comparative genomic analysis of the T6SS in sequenced strains has provided 179 information for understanding the distribution and importance of T6SS within species, 180 online bioinformatics databases have been used to annotate homologs of T6SS 181 automatically (e.g. SecretEPDB). 78 Bioinformatics platforms provide a resource for 182 potential effectors using conserved domains/motifs as queries in the databases. 79,80 183 Searching for conserved signaling peptides using bioinformatics tools normally 184 ameliorates putative effector discovery. Unfortunately, high diversity and the lack of a 185

Conclusions 238
Scientific ideology that the T6SS is largely antibacterial has been shifted, as we 239 demonstrate in this review, and recently by others. 87 Our challenge, however, is that not 240 only is the T6SS versatile, it is also complicated. Because of its divergent characteristics, 241 there is an accompanying lack of a standard regulatory mechanism or effector repertoire 242 and conduct for the system. This is because each T6SS assumes a certain uniqueness 243 to the next T6SS owing to the structural, target, and mode of action by a diversity of 244 effectors. Therefore, we are yet to make captivating discoveries using analytical methods 245 to study T6SS virulence effector proteins in relation to growth inhibition in eukaryotes. To 246 understand the mechanism and the biological significance of any secretion system, its 247 secreted proteins need to be identified and characterized. 52 This information will enable 248 us to set goals post research findings and strategies for translation of these outputs. 249 Pathogens are studied so that we can either control or exploit them. 250 There are several possibilities to explore once T6SS effectors targeting eukaryotic hosts 251 such as plants are identified. The T6SS occupies an intersection between eukaryotes, 252 prokaryotes, and the environment, therefore any contribution to the information pool about 253 this system may lead to development of animal, human and crop disease management 254 strategies. In plants, identified effectors that are recognised by resistance or avirulent 255 proteins may be used to identify resistance genes in distant relatives of non-hosts. These 256 can then be expressed in susceptible targets to confer protection. Protein-protein and 257 structural studies can show how the effector and the target interact. Effector-target 258 binding sites can be manipulated to render them non-targets without affecting functions 259 that could be beneficial to the host cell. 88 260 A great deal has been achieved in understanding the operating principles of the T6SS 261 and its secreted proteins. However, there is still a lot we do not know about the intricate 262 dialogues it mediates between different bacteria, eukaryotic host, and their respective 263 environments. Moreover, prediction of putative effectors may be improved by coupling 264 machine-learning programs with in vivo expression data, as seen with the prediction of 265 effectors from fungal secretomes. 89 In the foreseeable future, machine learning will be 266 the mainstream tool for effector discovery.