Genetic evidence for a regulated cysteine protease catalytic triad in LegA7, a Legionella pneumophila protein that impinges on a stress response pathway.

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate MAPK activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on high osmolarity medium. Mutations were isolated in the two amino-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two amino-terminal ankyrin repeat regions that modulate the function of the catalytic domain.

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila.

IMPORTANCE: Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBSTLp) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBSTEc). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipTLp) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.

F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.

Mobile Element Integration Reveals a Chromosome Dimer Resolution System in Legionellales.

In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The dimers must be resolved before cell separation to ensure genomic stability and cell viability. Dimer resolution is achieved by the broadly conserved dif/Xer system, which catalyzes one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here, we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (here named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Finally, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids. IMPORTANCE The maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers after they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif within the same molecule, leading to rapid site-specific recombination and conversion back into chromosome monomers. The apparent absence of chromosome dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens.

Legionella pneumophila CRISPR-Cas Suggests Recurrent Encounters with One or More Phages in the Family Microviridae.

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires' disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella mobile element 1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila's adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants-all targeted by previously and newly identified CRISPR-Cas spacers-but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae. These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires' disease is an often-fatal pneumonia caused by Legionella pneumophila, which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the "missing" L. pneumophila phages belong to the microvirus subfamily Gokushovirinae. Gokushoviruses are known to infect another intracellular pathogen, Chlamydia. How do gokushoviruses access L. pneumophila (and Chlamydia) inside their "cozy niches"? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

Draft Genome Sequence of Staphylococcus chromogenes ATCC 43764, a Coagulase-Negative Staphylococcus Strain with Antibacterial Potential.

Staphylococcus chromogenes can cause subclinical mastitis in cows, and some strains have also demonstrated antibacterial activity against pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we report the draft genome sequence of the S. chromogenes type strain ATCC 43764, which secretes the prodrug 6-thioguanine (6-TG), which antagonizes MRSA virulence.

Coagulase-negative staphylococci release a purine analog that inhibits Staphylococcus aureus virulence.

Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.

Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila.

In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system's array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.

Identification and characterization of a large family of superbinding bacterial SH2 domains.

Src homology 2 (SH2) domains play a critical role in signal transduction in mammalian cells by binding to phosphorylated Tyr (pTyr). Apart from a few isolated cases in viruses, no functional SH2 domain has been identified to date in prokaryotes. Here we identify 93 SH2 domains from Legionella that are distinct in sequence and specificity from mammalian SH2 domains. The bacterial SH2 domains are not only capable of binding proteins or peptides in a Tyr phosphorylation-dependent manner, some bind pTyr itself with micromolar affinities, a property not observed for mammalian SH2 domains. The Legionella SH2 domains feature the SH2 fold and a pTyr-binding pocket, but lack a specificity pocket found in a typical mammalian SH2 domain for recognition of sequences flanking the pTyr residue. Our work expands the boundary of phosphotyrosine signalling to prokaryotes, suggesting that some bacterial effector proteins have acquired pTyr-superbinding characteristics to facilitate bacterium-host interactions.

Discovery of Ubiquitin Deamidases in the Pathogenic Arsenal of Legionella pneumophila.

Legionella pneumophila translocates the largest known arsenal of over 330 pathogenic factors, called "effectors," into host cells during infection, enabling L. pneumophila to establish a replicative niche inside diverse amebas and human macrophages. Here, we reveal that the L. pneumophila effectors MavC (Lpg2147) and MvcA (Lpg2148) are structural homologs of cycle inhibiting factor (Cif) effectors and that the adjacent gene, lpg2149, produces a protein that directly inhibits their activity. In contrast to canonical Cifs, both MavC and MvcA contain an insertion domain and deamidate the residue Gln40 of ubiquitin but not Gln40 of NEDD8. MavC and MvcA are functionally diverse, with only MavC interacting with the human E2-conjugating enzyme UBE2N (Ubc13). MavC deamidates the UBE2N∼Ub conjugate, disrupting Lys63 ubiquitination and dampening NF-κB signaling. Combined, our data reveal a molecular mechanism of host manipulation by pathogenic bacteria and highlight the complex regulatory mechanisms integral to L. pneumophila's pathogenic strategy.

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways.

Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called "effectors" into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins are the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase-hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPK-controlled pathways in both yeast and human cells. Our findings indicate that L. pneumophila's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.

From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella.

The 1976 outbreak of Legionnaires' disease led to the discovery of the intracellular bacterial pathogen Legionella pneumophila. Given their impact on human health, Legionella species and the mechanisms responsible for their replication within host cells are often studied in alveolar macrophages, the primary human cell type associated with disease. Despite the potential severity of individual cases of disease, Legionella are not spread from person-to-person. Thus, from the pathogen's perspective, interactions with human cells are accidents of time and space-evolutionary dead ends with no impact on Legionella's long-term survival or pathogenic trajectory. To understand Legionella as a pathogen is to understand its interaction with its natural hosts: the polyphyletic protozoa, a group of unicellular eukaryotes with a staggering amount of evolutionary diversity. While much remains to be understood about these enigmatic hosts, we summarize the current state of knowledge concerning Legionella's natural host range, the diversity of Legionella-protozoa interactions, the factors influencing these interactions, the importance of avoiding the generalization of protozoan-bacterial interactions based on a limited number of model hosts and the central role of protozoa to the biology, evolution, and persistence of Legionella in the environment.

Priming in a permissive type I-C CRISPR-Cas system reveals distinct dynamics of spacer acquisition and loss.

CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3'-5' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under 'priming' conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences - but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event - with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.

Genome-based Definition of an Inflammatory Bowel Disease-associated Adherent-Invasive Escherichia coli Pathovar.

BACKGROUND: Mucosal-associated Escherichia coli are commonly found in inflamed tissues during inflammatory bowel disease (IBD). These bacteria often possess an adherent and invasive phenotype but lack virulence-associated features of well-described intestinal E. coli pathogens, and are of diverse serology and phylotypes, making it difficult to correlate strain characteristics with exacerbations of disease. METHODS: The genome sequences of 14 phenotypically assigned adherent-invasive Escherichia coli (AIEC) isolates obtained from intestinal biopsies of patients with IBD were compared with the genome sequences of 37 other pathogenic and commensal E. coli available from public databases. RESULTS: Core genome-based phylogenetic analyses and genome-wide comparison of genetic content established the existence of a closely related cluster of AIEC strains with 3 distinct genetic insertions differentiating them from commensal E. coli. These strains are of the B2 phylotype have a variant type VI secretion system (T6SS-1), and are highly related to extraintestinal pathogenic E. coli, suggesting that these 2 clinically distinct pathovars have common virulence strategies. Four other mucosally adherent E. coli strains from patients with IBD were of diverse phylogenetic origins and lacked the 3 genetic features, suggesting that they are not related to the B2 AIEC cluster. Although AIEC are often considered as having a unique association with Crohn's disease, isolates from Crohn's disease and ulcerative colitis were genetically indistinguishable. CONCLUSIONS: B2 AIEC thus represent a closely related cluster of IBD-associated E. coli strains that are distinct from normal commensal isolates, and which should be considered separately from the phenotypically similar but genetically distinct non-B2 AIEC strains when considering their association with intestinal pathogenesis.

Legionella pneumophila, armed to the hilt: justifying the largest arsenal of effectors in the bacterial world.

Many bacterial pathogens use dedicated translocation systems to deliver arsenals of effector proteins to their hosts. Once inside the host cytosol, these effectors modulate eukaryotic cell biology to acquire nutrients, block microbial degradation, subvert host defenses, and enable pathogen transmission to other hosts. Among all bacterial pathogens studied to date, the gram-negative pathogen, Legionella pneumophila, maintains the largest arsenal of effectors, with over 330 effector proteins translocated by the Dot/Icm type IVB translocation system. In this review, I will discuss some of the recent work on understanding the consequences of this large arsenal. I will also present several models that seek to explain how L. pneumophila has acquired and subsequently maintained so many more effectors than its peers.

Crystal structure of the Legionella pneumophila Lem10 effector reveals a new member of the HD protein superfamily.

Legionella pneumophila, the intracellular pathogen that can cause severe pneumonia known as Legionnaire's disease, translocates close to 300 effectors inside the host cell using Dot/Icm type IVB secretion system. The structure and function for the majority of these effector proteins remains unknown. Here, we present the crystal structure of the L. pneumophila effector Lem10. The structure reveals a multidomain organization with the largest C-terminal domain showing strong structural similarity to the HD protein superfamily representatives. However, Lem10 lacks the catalytic His-Asp residue pair and does not show any in vitro phosphohydrolase enzymatic activity, typical for HD proteins. While the biological function of Lem10 remains elusive, our analysis shows that similar distinct features are shared by a significant number of HD domains found in Legionella proteins, including the SidE family of effectors known to play an important role during infection. Taken together our data point to the presence of a specific group of non-catalytic Legionella HD domains, dubbed LHDs, which are involved in pathogenesis.

Molecular Characterization of LubX: Functional Divergence of the U-Box Fold by Legionella pneumophila.

LubX is part of the large arsenal of effectors in Legionella pneumophila that are translocated into the host cytosol during infection. Despite such unique features as the presence of two U-box motifs and its targeting of another effector SidH, the molecular basis of LubX activity remains poorly understood. Here we show that the N terminus of LubX is able to activate an extended number of ubiquitin-conjugating (E2) enzymes including UBE2W, UBEL6, and all tested members of UBE2D and UBE2E families. Crystal structures of LubX alone and in complex with UBE2D2 revealed drastic molecular diversification between the two U-box domains, with only the N-terminal U-box retaining E2 recognition features typical for its eukaryotic counterparts. Extensive mutagenesis followed by functional screening in a yeast model system captured functionally important LubX residues including Arg121, critical for interactions with SidH. Combined, these data provide a new molecular insight into the function of this unique pathogenic factor.

Silencing by H-NS potentiated the evolution of Salmonella.

The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in Salmonella, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in Salmonella. Six independently derived S. Typhimurium hns mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the hns mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the Salmonella Pathogenicity Island-1 (SPI-1) Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in Salmonella hns mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type Salmonella lineages did not acquire these mutations. The stpA missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.