Unprecedented Protein Divergence within a T3SS Family

Selection pressure drives rapid emergence of antibiotic resistance mechanisms promoting searches for therapeutic targets in bacterial processes needed for virulence, not viability, which include the Type Three Secretion System (T3SS). Distinct T3SS families evolved from the flagellar export apparatus where remaining homology hinders development of anti-T3SS specific therapies. Around 15 proteins that are highly-conserved within, but not between T3SS families yet such divergence is rarely leveraged, to promote understanding, due to unknown evolutionary histories. Here we document unprecedented divergence in two ‘LEE’ T3SS family members. Interchangeability studies uncover unusual LEE biology (eg 2-orf genes) and illustrate each T3SS protein can tolerate dramatic change. Functional defects (12 proteins) and novel phenotypes enabled studies that reveal i) pathotype-specific protein functionality, ii) T3SS crosstalk with other processes, and iii) potential therapeutic targets. The work provides resources and testable predictions for further discoveries and will promote comparable studies between distinct T3SS families.


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The action of antibiotics that target physiologically important bacterial processes such as 26 flagellar movement or cell wall biosynthesis are rapidly undermined by the evolution of 27 resistance mechanisms (Lyons and Strynadka, 2019;Roope et al., 2019). As stated by the 28 World Health Organisation (2021) "Antibiotic resistance is one of the biggest threats to global 29 health, food security, and development today.". There is an emerging interest in identifying 30 therapeutic targets against processes important for virulence, but not viability, such as Type Most knowledge on T3SS protein functionality relates to studies on prototypic systems 50 from 5 families where homologs share as little as 21% similarity (Diepold and Wagner, 2014). 51 Such divergence argues for selection pressure driving changes beneficial to the host bacteria's 52 lifecycle leading to a pathotype-specific T3SS. While such divergence provides opportunities 53 for interrogating T3SS protein biology and developing anti-T3SS (broad-or pathotype-54 specific) therapeutics, it is rarely leveraged due to unknown evolutionary histories (Klein et al.,55 2017). 56 Here we describe how our interest in an apparently degenerate LEE region led to the 57 uncovering of two, overlooked, examples of unprecedented divergence within an evolutionary 58 closely-related T3SS family. We reveal this region is not degenerate but has unusual biology 59 while studies with the homologs i) undermine the idea of high LEE T3SS protein conservation 60 reflecting functional constraints, ii) illustrate many, if not all the T3SS proteins possess 61 pathotype-specific functionality, iii) reveal T3SS crosstalk with other processes, iv) provide a 62 strategy for identifying T3SS-and/or pathotype-specific therapeutic targets, v) forward testable 63 predictions and vi) describe phenotypes and tools to enable future discoveries.

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A majority of tarda LEE T3SS proteins functionally replace their EPEC counterparts 82 Such unprecedented divergence prompted assessment of whether the tarda genes could 83 functionally substitute their EPEC counterpart. Thus, tarda genes were PCR-cloned and 84 introduced into the appropriate EPEC T3SS gene-deficient strain to assess T3SS functionality 85 using two well-established assays (Kenny et al., 1997a;Kenny et al., 1997b). Strain genotype 86 was routinely supported by PCR analysis (not shown).

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Disrupting orf1 (not shown) compromised the export process unlike HA-tagging Orf2 ( Figure   101 2G) supporting independent Orf1/Orf2 expression. This work illustrates that tarda LEE 2-orf 102 genes can produce functional proteins but perhaps not via predict ribosome-mediated 103 mechanisms.

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The demonstration of 27 LEE tarda (includes four 2-orf) genes producing full-length 136 and/or functional proteins suggests the complementation defects are not due to non-expression 137 but reduced expression or, more likely, sequence divergence. To explore this hypothesis studies 138 focused on the final 'complementation-null' group member, EscV as in highest homology 139 category (66.3% identity; Figure 1B and S3_v). The EscV C-terminal domain provides the 140 T3SS export gate and SepL docking sites (Gaytan et al., 2018;Portaliou et al., 2017).

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Divergence within multiple docking sites ( Figure 4A) prompted exchanging the entire C-142 terminal region for that from EPEC generating a chimera that rescued all export defects ( Figure   143 4B and 4C) clearly linking the defects to divergence.  Figure 5D) implicated divergence in the final 54 residues prompting a focus on the most 162 divergent area: residues 128-137 (0% identity; Figure 5C). Swapping the 10 tarda residues for 163 EPECs 9 generated a chimera (C5) restored significant levels of Tir delivery and Tir-Intimin 164 interaction ( Figure 5C) reporting a key role for this with other, undefined, C-terminal features.

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Replacing the final tarda 8 residues for EPEC's increased Tir delivery levels ( Figure 6B i.e. two non-conservative changes ( Figure 6C). Surprisingly, the EspDmulti and triple-178 substituted variants had predicted C-terminal CCFs ( Figure 6D) and, whilst absent from tarda 179 EspD, could readily be generated by changing two residues to their EPEC counterpart (  Pathotype-specific T3SS protein functionality 191 To further support that phenotypes arising from expressing a variant (EPEC,tarda,chimera) 192 can be leveraged to describe pathotype-specific functionality, studies were undertaken on a 193 ΔsepL mutant phenotype. The latter refers to observing faster migration of proteins through 194 SDS-PA gels (most evident for those >40kDa) with this phenotype rescued by re-introducing 195 EPEC, not tarda sepL ( Figure 7A). Screening virulence factor-deficient strains revealed the  Uncovering two examples of unprecedented divergence within an evolutionarily closely-237 related T3SS family has led to the discovery i) that all LEE T3SS proteins can tolerate dramatic 238 change, ii) of tools and strategies to identify therapeutic targets and cryptic, pathotype-specific, 239 functionality, and iv) of novel LEE biology. Moreover, the work provides unique resources 240 and testable predictions with implications for other T3SS families and the identification of anti-241 virulence (broad-or pathotype-specific) targets. to the strain's lifecycle leading to a pathotype-specific T3SS.