Integrating signals to drive type VI secretion system killing

1 Integrating signals to drive T6SS killing 2 3 Patricia Bernal1, Marina Murillo-Torres1,2, Luke P. Allsopp2* 4 5 6 1Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Seville, 7 41012, Spain 8 2National Heart and Lung Institute, Imperial College London, London, UK 9 10 *For correspondence. Email l.a[email protected]; Tel. (+44) 020 7594 7829 11 12 13 The type VI secretion system (T6SS) is a nanoweapon used by bacteria to defend from 14 predators, subvert eukaryotic cells and fight other microbes including bacterial 15 competitors and fungi (Allsopp et al., 2020). Functioning like a spring-loaded harpoon, 16 bacteria fire this system directly into target cells directly delivering a toxic payload for 17 bacterial gain (Allsopp et al., 2020). Identified in more than 25% of Gram-negative 18 bacteria and containing 13 core components, the T6SS has found widespread use in 19 both environmental and pathogenic organisms (Allsopp et al., 2020). The core 20 components are typically encoded in one large operon but orphan gene clusters 21 encoding additional components are common. For instance, Vibrio cholerae strains 22 contain one large central cluster and between 2 to 5 auxiliary (or orphan) islands 23 (recently reviewed in Crisan and Hammer, 2020). 24 The high conservation of the large clusters encoding the T6SS and orphan islands 25 indicates that these systems are expressed and confer a selective advantage to the 26 bacteria. However, the expression, assembly and secretion of the many proteins 27 required to form a functional T6SS is energetically costly and may not be necessary or 28 favourable for all environments and/or conditions encountered by a bacterium (Basler, 29 2015). Indeed, unless resources are limited or cell density is high it makes little sense to 30 expend resources being highly aggressive (Gonzalez and Mavridou, 2019). Thus, 31 expression of the T6SS is highly regulated in many organisms, presumably to enable 32 expression in defined conditions (Silverman et al., 2012; Allsopp et al., 2017; Wang et 33 al., 2019). 34 Control of the T6SS occurs at multiple levels with complex transcriptional (e.g. VasH), 35 post-transcriptional (e.g. RsmA) and post-translational (e.g. PpkA/Fha) regulatory 36 mechanisms present in bacteria (Figure 1) (Silverman et al., 2012; Allsopp et al., 2017; 37 Wang et al., 2019). Many T6SS genes are not expressed well in laboratory conditions, 38 however a common theme is enhanced expression in the later stages of growth, nutrient 39 availability and upon surface contact (Silverman et al., 2012; Wang et al., 2019). 40 Moreover, different bacteria have integrated T6SS genes into a variety of existing 41 regulatory networks enabling expression under beneficial conditions. Major drivers of the 42 T6SS are; Quorum sensing, salinity, osmolality, metal iron availability and stress 43 (Silverman et al., 2012; Wang et al., 2019; Crisan and Hammer, 2020). Some of these 44 conditions are sensed by bacteria via two-component systems and relayed into 45 transcriptional regulators for direct control of T6SS genes. However, others act indirectly 46 and for most of them the mechanism is still unclear or complex with multiple regulators 47 at play. 48 Regardless of the regulators at play, transcription is the first critical step and requires 49 RNA polymerase for mRNA synthesis to occur. Sigma factors specify transcription by 50 binding to characteristic promoter sequences, recruiting the RNA polymerase and 51 enabling the formation of the open complex (Browning and Busby, 2004). The expression 52 of the majority of genes is controlled by the ‘housekeeping’ sigma factor 70 (also known 53 as RpoD, σ70 σA) (Browning and Busby, 2004). However, the major alternative sigma 54 factor, RpoN (Sigma 54, σ54 and σN), has been implicated in modulating the T6SS in 55 many Gram-negative bacteria (Bernard et al., 2011; Silverman et al., 2012; Crisan and 56 Hammer, 2020; Seibt et al., 2020). 57 RpoN was originally discovered to be a central player in controlling nitrogen metabolism 58 (Francke et al., 2011). It is also a key factor driving flagella biosynthesis and motility 59 (Francke et al., 2011). More recently, this regulator has been shown to be a general 60 regulator of virulence, and numerous bacterial cell envelope and surface components 61 (Francke et al., 2011). Unlike Sigma 70, RpoN requires an enhancer binding protein 62 (EBP) for activation. EBPs typically bind within 100-200 bp of an RpoN binding site; DNA 63 bending via integration host factor (IHF) enables the interaction between the EBP and 64 RpoN leading to open complex formation and transcription (Schumacher et al., 2006). 65 Without the EBP, RpoN binds and prevents transcription from a locus (Schaefer et al., 66 2015). EBPs are typically comprised of three domains; an N-terminal regulatory domain, 67 an AAA+-family ATPase domain, and a C-terminal Helix-turn-Helix DNA-binding domain 68 that recognises the -24 and -12 promoter elements (Schumacher et al., 2006). Variability 69 in the N-terminal and C-terminal domains amongst EBPs enable a diverse range of 70 signals to be detected and distinct DNA sequences to be bound (Francke et al., 2011). 71 Therefore, influencing the availability of EBPs and their stimuli enable the bacteria to 72 respond to their environment by modulating different surface components including the 73 T6SS. 74 RpoN and EBPs are involved in the regulation of T6SS genes in Vibrio cholerae, 75 Pseudomonas aeruginosa, and Klebsiella pneumoniae amongst others (Bernard et al., 76 2011; Sana et al., 2013; Storey et al., 2020). In most cases, RpoN is critical for the 77 expression of the T6SS, but it has been reported to play an indirect repressive role in the 78 regulation of the H2-T6SS of P. aeruginosa (Bernard et al., 2011; Sana et al., 2013; 79 Storey et al., 2020). Interestingly, most EBPs associated with the T6SS are encoded 80 within the central T6SS clusters (Bernard et al., 2011; Sana et al., 2013). This is the case 81 for Sfa2 and Sfa3, two RpoN enhancer proteins of P. aeruginosa that are encoded within 82 the H2- and H3-T6SS clusters, respectively (Sana et al., 2013). Similarly, PSPTO_2549 83 and PSPTO_5424, found within the HIS-I and HIS-II T6SS clusters of P. syringae pv. 84 tomato, encoded two putative σ54 transcriptional regulators (Chien et al., 2020) with 57% 85 and 71% identity to Sfa2. Certainly, the best characterised T6SS-related EBP is VasH, 86 a σ54 activator necessary for functional T6SSs in Aeromonas hydrophila, Vibrio fischeri, 87 Pectobacterium atrosepticum, Marinomonas sp. and most importantly V. cholerae 88 (Bernard et al., 2011; Kitaoka et al., 2011; Guckes et al., 2020). 89 In V. cholerae strain O1 El Tor A1552, an isolate that causes cholera disease, vasH is 90 encoded within the central cluster together with the majority of the system components 91 (Pukatzki et al., 2006). VasH is required for the RpoN-dependent transcription of two 92 orphan islands in this strain (Kitaoka et al., 2011). These orphan operons contain the hcp 93 and vgrG genes, which encode for the tube and the tip proteins of the system, two vital 94 core components required for a functional T6SS (Zheng et al., 2011). Interestingly, in 95 A1552 the production of Hcp and VgrG is only detected under low temperature and high 96 osmolarity conditions whilst the structural cluster has a higher transcription rate (Ishikawa 97 et al., 2012). Recently, the VasH-dependent molecular mechanism integrating the 98 environmental cues that co-regulate the structural and the orphan clusters of A1552 have 99 been explored by Seibt et al. (2020). 100 101 Since the activity of EBPs are normally controlled by their N-terminal domains 102 (Schumacher et al., 2006; Francke et al., 2011), Seibt et al. expressed a VasHA1552 103 truncated version lacking the N-terminal regulatory domain which resulted in a 104 constitutively-active T6SS that was independent of temperature and osmolarity (Seibt et 105 al., 2020). In line with a previous report, VasHA1552 was not necessary for the expression 106 of the central large structural operon within which it is encoded (Dong and Mekalanos, 107 2012). Rather, VasH acts to bind the promoters and switch-on expression of the two hcp 108 and vgrG orphan clusters in conjunction with RpoN and IHF (Seibt et al., 2020). Thus, 109 the environmental cues regulating the structural cluster are integrated into the expression 110 of the orphan clusters through VasH, in a sophisticated mechanism of cascading 111 regulation. In this mechanism, VasH acts as a gatekeeper. VasH is expressed with the 112 main cluster under conditions of low temperature and high osmolarity. Once VasH levels 113 reached those required to activate transcription, the gate is opened and expression of 114 the orphan operons occurs. This results in production of the missing core components 115 of the T6SS (Hcp and VgrG) leading to a functional and finely tuned apparatus. 116 Contrary to the complexity of the T6SS regulation in A1552, the less virulent V52, which 117 causes gastrointestinal irregularities, displays constitutive and signal independent 118 expression of the T6 system (Pukatzki et al., 2006). This means that strains of V. 119 cholerae with different degrees of virulence in humans have distinct T6SS expression 120 strategies with V52 being offensive and continuously expending energy firing its T6SS, 121 whilst A1552 opts for a defensive strategy. This example illustrates how bacteria use a 122 sophisticated regulatory network to integrate signalling cues from the environment and 123 orchestrate a defensive plan when necessary. 124 In other bacteria, the environmental cues regulating T6SSs are known, although the fine 125 regulatory network behind it may not be clear. For instance, the T6SS of A. tumefaciens 126 is induced under acidic pH conditions like those encountered by the bacterium upon leaf 127 wounding and in the apoplast when they penetrate the plant (Wu et al., 2012). The T6SSs 128 of opportunistic enteric pathogen Edwardsiella tarda and enteroaggregative E. coli 129 (EAEC) are repressed in the presence of iron through the action of the regulator Fur 130 (Bernard et al., 2011). Some bacteria can also sense killing of their kin, such as P. 131 aeruginosa that recognises specific signals released from lysed Pseudomonas to 132 activate a danger sensing mechanism that promotes T6SS production through the 133 GacA/S cascade (Le Roux et al., 2015). In other cases, bacteria respond to cues from 134 the host environment. For example, V. cholerae induces the T6SS in the presence of 135 chitin, the main component of the crustaceans exoesqueletons they colonise, killing 136 competitors on the host and even up taking their DNA (Borgeaud et al., 2015). Similarly, 137 V. fischeri upregulates the expression of the T6SS upon colonisation of the squid light 138 organ to be the sole resident (Speare et al., 2018). In a few cases, the specific host signal 139 has been identified e.g. the mammalian mucin from gut secretion activates T6SS in V. 140 cholerae and allows this strain to fight microbiome competitors (Bachmann et al., 2015). 141 In a similar strategy, the T6SS of Klebsiella pneumoniae is transcriptionally induced by 142 polymyxin antibiotics and the antimicrobial peptide human β-defensin 3 via the action of 143 the two-component system PhoPQ (Storey et al., 2020). Thus, bacteria can ‘wire-up’ the 144 regulator control of the T6SS to respond to their environment optimally. 145 T6SS regulation can be highly complex with multiple layers of control to secure an 146 optimal balance between defence, aggression and energy expenditure. The number of 147 environmental cues responsible for controlling the T6SSs is continuously growing but for 148 many of the systems they are still unknown. The integration of environmental cues 149 through VasH is a formidable example of successful evolution of a control mechanism 150 for an “expensive” but advantageous system to drive the killing of a bacterium’s 151 competitors. 152 153 Acknowledgements 154 Work in the L.P.A laboratory is facilitated by a Research Grant 2020 from the European 155 Society of Clinical Microbiology and Infectious Diseases to LPA as well as funds from 156 the National Heart and Lung Institute at Imperial College London and in an environment 157 that received funds from the Cystic Fibrosis Trust to establish the Strategic Research 158 Centre: Personalised approach to Pseudomonas aeruginosa (PAPA). Work in P.B 159 laboratory is facilitated by the "Research Challenges" 2018 R+D+i Project RTI2018- 160 096936-J-I00 grant funded by the Spanish Ministry of Science, Innovation and 161 Universities. 162 163 References 164 Allsopp, L.P., Bernal, P., Nolan, L.M., and Filloux, A. 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