A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation

Arden Perkins, Dan A. Tudorica, Raphael D. Teixeira, Tilman Schirmer, Lindsay Zumwalt, O. Maduka Ogba, C. Keith Cassidy, Phillip J. Stansfeld, Karen Guillemin
2020
19 The reactive oxygen species produced during inflammation through the neutrophilic respiratory 20 burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among 21 these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and 22 strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. 23 Thus, HOCl can be viewed as a host-specific cue that conveys important information about what 24
more » ... bout what 24 bacterial physiology and lifestyle programs may be required for successful colonization. 25 Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly 26 selective detection of HOCl due to its reactive and transient nature and chemical similarity to more 27 benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in 28 response to increasing HOCl levels E. coli regulates biofilm production via activation of the 29 diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific 30 oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-31 : bioRxiv preprint 2 binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation 32 by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum 33 mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state 34 alters the delicate equilibrium of competition for Zn ++ between the CZB domain and other zinc 35 binders to relay the presence of HOCl through activating the associated GGDEF domain to 36 catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the 37 conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the 38 biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated 39 diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent 40 and are possessed by pathogens that manipulate host inflammation as part of their colonization 41 strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved 42 and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal 43 common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the 44 host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases 45 of chronic inflammation and gut dysbiosis. 46 47 a bactericide through the oxidation of a broad spectrum of cellular components, especially sulfur-53 containing amino acids 3-5 . However, only trace amounts of HOCl form in the absence of enzymatic 54 catalysis, and so HOCl also represents a unique chemical cue that signals the presence of an animal 55 host. The ability to detect HOCl in the environment can allow bacteria to optimize their lifestyle 56 for host colonization and/or enduring host inflammation; but achieving high selectivity and 57 sensitivity of a reactive and labile chemical is not an easy task. A second challenge is that other 58 chemically-similar oxidants exist, such as hydroperoxides (ROOH), that do not necessarily convey 59 the same information about a bacterium's environment. For example, hydrogen peroxide (H2O2), 60 which bacteria are well-equipped to eliminate 6,7 , is far less toxic 8 and is produced through many 61 mechanisms other than immune responses, such as bacterial metabolism 9 , and so its presence is 62 : bioRxiv preprint not host-specific per se. Thus, the detection of HOCl requires a sensing apparatus to overcome the 63 oxidant's propensity for non-specific oxidation and to distinguish between other oxidants that can 64 react with a similar suite of molecular targets. In fact, synthetic molecular probes have only 65 recently overcome this challenge 10,11 . 66 We recently identified a novel bacterial sensing system in which a chemoreceptor zinc-67 binding (CZB) protein domain detects the inflammation product HOCl through direct oxidation of 68 a reactive cysteine to form cysteine sulfenic acid (Cys-SOH, Fig. S1 ) 12,13 . The molecular 69 mechanism is thought to accomplish selective reactivity with HOCl through a conserved zinc-70 thiolate switch 12,13 , a chemical moiety with enhanced reactivity toward HOCl 5,14,15 . As an example 71 of the biological significance of this mechanism and relevance to human disease, CZB sensing of 72 HOCl was shown to facilitate chemoattraction to HOCl sources for the gastric pathogen H. pylori 73 through regulation of the chemoreceptor transducer-like protein D (TlpD), providing an 74 explanation for the bacteria's persistence in inflamed tissue and tropism for gastric wounds 12,16-18 . 75 Observations of other CZB-containing Gammaproteobacteria that can inhabit inflamed 76 environments, such as Salmonella enterica 19 and Escherichia coli 20 , prompted us to investigate 77 whether sensing of host HOCl by CZB proteins could have broad utility in regulating bacterial 78 lifestyles critical for host colonization. 79 CZB domains remain poorly characterized, but CZB-containing proteins are reported to 80 play roles in sensing exogenous zinc, pH, and oxidants to regulate bacterial chemoreceptors and 81 diguanylate cyclases 12,21-25 . Earlier work has shown CZBs to be approximately 15 kD in size and 82 consist of a four-helix bundle fold with a unique and conserved 3His/1Cys zinc-binding motif 23,26 , 83 with the cysteine of this motif stabilized as a thiolate and activated to be oxidized by HOCl 13 . Prior 84 to this study, it was unknown by what molecular mechanism CZB domains could integrate signals 85 from such divergent ligands as HOCl and Zn ++ into cellular responses. Cellular zinc homeostasis 86 involves a complex equilibrium between many high affinity zinc binders that compete for Zn ++ 87 and maintain the cytosolic Zn ++ concentration near zero 27,28 , and CZB affinity for zinc is thought 88 to be in the sub-femtomolar range 23 . In the highly competitive environment of the cell cytosol even 89 small changes in a protein's zinc affinity can dramatically shift the binding equilibrium between 90 zinc-bound and zinc-free states 27-30 . Thus, we sought to study whether alterations to the CZB zinc-91 binding core through HOCl oxidation might regulate the domain through influencing zinc binding. 92 However, a critical barrier to investigating this hypothesis, both in vitro and in vivo, is the transient 93 : bioRxiv preprint 4 nature of the Cys-SOH reaction intermediate, which can spontaneously reduce or become further 94 oxidized to cysteine-sulfinate (Cys-SO2 -) in the presence of excess oxidant 31,32 . So far, in vivo 95 studies on the biological roles of CZBs have mostly relied on full-knockouts 12,16,21,23,33 and the 96 importance of the conserved zinc-binding cysteine has not previously been investigated in vivo. 97 The ubiquitous bacterial signaling molecule bis-(3′-5′)-cyclic dimeric guanosine 98 monophosphate (c-di-GMP) is well-known to play a pivotal role in bacterial decisions of cell 99 adhesion and biofilm formation, that in turn have relevance for the pathogenicity of many bacteria 100 involved in human diseases 34-36 . The existence of CZB-regulated diguanylate cyclases, which 101 catalyze the production of c-di-GMP from guanosine triphosphate (GTP), prompted us to ask if c-102 di-GMP signaling processes, such as biofilm formation, can be regulated in response to HOCl, and 103 thus, to host inflammation. To address this question, we have for the first time quantified CZB 104 conservation patterns to understand the underlying functional rationale as it pertains to ligand-105 sensing and signal transduction, determined the prevalence of CZB-containing protein 106 architectures to identify the molecular pathways they regulate, and documented the full biological 107 distribution of these proteins to learn the breadth of environments and organisms in which these 108 proteins operate. These data reveal the E. coli diguanylate cyclase Z (DgcZ, previously referred to 109 as YdeH 23,37-39 ) as an exemplar CZB-regulated diguanylate cyclase representative of a large subset 110 of CZB-containing proteins, and we have utilized DgcZ as a model system to obtain broad insight 111 into CZB HOCl-sensing and regulation of bacterial biofilm formation. 112 We find that E. coli DgcZ, is highly and preferentially reactive with biologically-relevant 113 concentrations of HOCl and regulates c-di-GMP catalysis and cellular biofilm through direct 114 oxidation of the zinc-binding cysteine. Biofilm and surface attachment is increased by micromolar 115 HOCl through DgcZ and can also be induced by strains harboring a DgcZ point mutant that mimics 116 cysteine oxidation. These data support earlier reports that oxidants can increase E. coli biofilm 117 formation 24,40 and provide new insight into how enteropathogenic, enteroaggregative, and 118 uropathogenic E. coli (EPEC, EAEC, UPEC) may respond to host inflammation to favor 119 pathogenicity 41-43 . We propose a new unifying model for how CZB proteins can facilitate both 120 HOCl and zinc-sensing, with relevance for bacterial biology across diverse phyla and human 121 : bioRxiv preprint 5 diseases of gut dysbiosis and chronic inflammation. The ability of CZB domains to selectively 122 sense HOCl implicates this family of proteins as bacterial inflammation sensors. 123 124 RESULTS 125 126 Architectures, conservation, and biological distribution of CZB-containing proteins 127 Protein structure-function relationships can be revealed through analyses of conservation patterns 128 to learn what parts of the protein are indispensable for function across divergent homologues 44-46 . 129 To create a database of all currently-known CZB-containing proteins we performed iterative 130 searches with the Basic Local Alignment Search Tool (BLAST) 47 for CZB domain sequences in 131 the non-redundant protein database, which resulted in 8,227 sequences that contain the unique 132 3His/1Cys CZB zinc-binding motif, with 22.7 % pairwise identity across all sequences. Most 133 CZB-containing protein sequences contain multiple protein domains, so to understand the major 134 cellular pathways regulated by CZB domains, we further categorized and quantified sequences 135 according to protein architecture. We found that CZB-containing proteins can be divided into 136 seven subgroups based on domain similarity, with the majority of sequences (85.8 %) involved in 137 two biological outputs, namely chemotaxis or c-di-GMP metabolism (Fig. 1A-B) . Some sequences 138 also appear to contain only a CZB domain with no other detectable protein domain sequence 139 signature (12.6 %), although some of these may represent incomplete sequences or annotations. 140 The most common subgroup consists of soluble CZB-regulated chemoreceptors similar in 141 structure to H. pylori TlpD (59.4 %), which we refer to here as "TlpD-like." CZB-regulated 142 nucleotide cyclases, including E. coli DgcZ, account for a smaller but widespread fraction of 143 sequences (6.3 %), which we refer to as "DgcZ-like." Less common, but involved in functionally-144 related processes, are CZB-regulated chemotaxis W (CheW, 1.8 %) and Glu-Ala-Leu (EAL) (0.6 145 %) proteins that transduce chemoreceptor signals and degrade c-di-GMP, respectively. Nearly all 146 CZB sequences are predicted to be cytosolic, with only 384 putative periplasmic CZB sequences 147 (approximately 4 %) identified. 148 Commonalities in amino acid conservation between distantly related CZB sequences could 149 point toward general functions of CZB domains, while differences between subgroups could 150 indicate evolutionary tuning to optimize ligand-sensing and signal transduction in specific settings. : bioRxiv preprint 6 crystal structure of the E. coli DgcZ CZB domain, which is a homodimer with each monomer 153 composed of five α-helices and one 310-helix (Fig 1C, PDB code: 3t9o) 23,48 . In addition to the 154 ubiquitous 3His/1Cys zinc-binding motif, two regions of global conservation across all CZB 155 domains were revealed (Fig. 1C) . First, the N-terminal α1 helix exhibits a modest degree of 156 conservation, with 10 positions that have sequence identity conservation in the range of 20-100 %. 157 This region (residues 1-30) constitutes a large portion of the homodimer interface (384 Å 2 of 1950 158 Å 2 total) that forms a two-fold symmetry axis, with residues packing against their homodimer 159 counterpart. Second, in addition to the universally-conserved zinc-binding Cys, many residues of 160 the α3 region exhibit a high degree of conservation (Fig. 1C) . This pattern of conservation in the 161 α1 and α3 regions occurs across all CZB subgroups, suggesting these two regions are of universal 162 importance for CZB function. One additional site of high conservation occurs in the diguanylate 163 cyclase subgroup, where a Trp residue, which resides three sites downstream of the conserved 164 zinc-binding His22, packs into the protein core against the zinc-binding site (Fig. 1C , noted with 165 Asterix). 166 Oxidation of the conserved zinc-binding Cys, located in α3, was previously reported to 167 induce structural changes in the CZB domain, whereby Cys-SOH formation promotes detachment 168 of the Cys from the zinc core and a local unfolding of the α2-α3 region 12 (Fig. S1 ). The structural 169 change upon disruption of the Cys thiolate-zinc interaction is also directly observed in the crystal 170 structure of a DgcZ Cys→Ala mutant (PDB: 4h54) 23 . Therefore, the high conservation of the α3 171 region could relate to CZB signal transduction. To further investigate the conservation of the α3 172 region, seqlogo plots were generated for the seven-residue motif containing the conserved Cys for 173 all CZB sequences and CZB architecture subgroups (Fig. 1D) . By studying the position and 174 interactions of each residue in the E. coli DgcZ CZB structure, putative roles and rationales for 175 conservation were inferred for each amino acid site as follows: Position 1 is approximately 100 % 176 conserved as a Cys, reflecting its absolute requirement for function. The Cys forms part of the 177 zinc-binding core, increases zinc affinity by an order of magnitude 23 , and can serve as a redox-178 sensor through direct oxidation in some CZB proteins 12 . Positions 2 and 5 are conserved as 179 residues that either contain a hydrophilic side chain or a small hydrophobic side chain that permit 180 exposure to solvent. Positions 3, 6, and 7 are conserved as bulky hydrophobic side chains that are 181 buried in the protein core and provide a thermodynamic driving force for the proper folding of the 182 motif. Position 4 is almost universally conserved as a Gly likely for the reason that a Cβ atom 183
doi:10.5451/unibas-ep78864 fatcat:c3qh2z5htzdjpeyw4ta52l3jly