Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole.

The Legionella pneumophila Sde family of translocated proteins promotes host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity was tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, prevented binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb resulted in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decayed quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.

Identification of Determinants that Allow Maintenance of High-Level Fluoroquinolone Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We here identify proteins that contribute to the fitness of FQR strains overexpressing three known RND systems using high-density insertion mutagenesis. Overproduction of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced LOS biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump overproduction, which does not affect the outer membrane pump component, was relatively tolerant to loss of these functions, consistent with outer membrane protein overproduction being the primary disruptive component. Surprisingly, overproduction of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overproduction, resulting in activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from peroxide stress. These results indicate that the RND outer membrane protein overproduction is compensated by cytoplasmic buffering and maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates. Importance: Acinetobacter baumannii is a pathogen that often causes multidrug resistant (MDR) infections in healthcare settings, presenting a threat to the efficacy of known therapeutic interventions. Fluoroquinolones such as ciprofloxacin are currently ineffective against a majority of clinical A. baumannii isolates, many of which express pumps that remove this antibiotic class from within the bacterium. Three of these pumps can be found in most clinical isolates, with one of the three often hyperproduced at all times. In this study we identify proteins that are necessary for the fitness of pump hyperproducers. The identified proteins are necessary to stabilize the outer membrane and allow the cytoplasm to tolerate the accumulation of ions as a consequence of excess pump activity. These results point to strategies for developing therapies that combine known antibiotics with drugs that target proteins important for survival of strains hyperexpressing efflux pumps.

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.

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

Streptococcus pneumoniae favors tolerance via metabolic adaptation over resistance to circumvent fluoroquinolones.

Streptococcus pneumoniae is a major human pathogen of global health concern and the rapid emergence of antibiotic resistance poses a serious public health problem worldwide. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage and has remained rare. Our data indicate that deleterious fitness costs in the mammalian host constrain the emergence of fluoroquinolone resistance both by de novo mutation and recombination. S. pneumoniae was able to circumvent such deleterious fitness costs via the development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, resulting in a fitness benefit during infection of mice treated with fluoroquinolones. These data suggest that the emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes are an important contributor to the evasion of antibiotic-mediated killing.IMPORTANCEThe increasing prevalence of antibiotic resistant bacteria is a major global health concern. While many species have the potential to develop antibiotic resistance, understanding the barriers to resistance emergence in the clinic remains poorly understood. A prime example of this is fluroquinolone resistance in Streptococcus pneumoniae, whereby, despite continued utilization, resistance to this class of antibiotic remains rare. In this study, we found that the predominant pathways for developing resistance to this antibiotic class severely compromised the infectious capacity of the pneumococcus, providing a key impediment for the emergence of resistance. Using in vivo models of experimental evolution, we found that S. pneumoniae responds to repeated fluoroquinolone exposure by modulating key metabolic pathways involved in the generation of redox molecules, which leads to antibiotic treatment failure in the absence of appreciable shifts in resistance levels. These data underscore the complex pathways available to pathogens to evade antibiotic mediating killing via antibiotic tolerance.

Identification of essential genes that support fitness of Acinetobacter baumannii efflux pump overproducers in the presence of fluoroquinolone.

Acinetobacter baumannii is a nosocomial pathogen often associated with multidrug resistance (MDR) infections. Fluoroquinolone resistance (FQR) due to drug target site mutations and elevated expression of RND drug transporters is common among clinical isolates. We describe here a CRISPRi platform that identifies hypomorphic mutations that preferentially altered drug sensitivity in RND pump overproducers. An sgRNA library against essential genes of A. baumannii was constructed with single and double nucleotide mutations that produced titratable knockdown efficiencies and introduced into multiple strain backgrounds. Other than nusG depletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing clinically relevant RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs causing hypersensitivity were predicted to result in outer membrane dysfunction, to which the AdeFGH overproducer appeared particularly sensitive. Depletions of either the outer membrane assembly BAM complex, LOS biogenesis proteins, or Lpt proteins involved in LOS transport to the outer membrane caused drug hypersensitivity in at least two of the three pump overproducers. On the other hand, depletions of translation-associated proteins, as well as components of the proton-pumping ATP synthase pump resulted in fitness benefits for at least two pump-overproducing strains in the presence of the drug. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the efficacy of drug resistance in efflux overproducers was enhanced by slowed translation or defects in ATP synthesis linked to the control of proton movement across the bacterial membrane.

The Sde phosphoribosyl-linked ubiquitin transferases protect the Legionella pneumophila vacuole from degradation by the host.

Legionella pneumophila grows intracellularly within the membrane-bound Legionella-containing vacuole (LCV) established by proteins translocated via the bacterial type IV secretion system (T4SS). The Sde family, one such group of translocated proteins, catalyzes phosphoribosyl-ubiquitin (pR-Ub) modification of target substrates. Mutational loss of the entire Sde family results in small defects in intracellular growth, making it difficult to identify a clear role for this posttranslational modification in supporting the intracellular lifestyle. Therefore, mutations that aggravate the loss of sde genes and caused intracellular growth defects were identified, providing a mechanistic connection between Sde function and vacuole biogenesis. These double mutants drove the formation of LCVs that showed vacuole disintegration within 2 h of bacterial contact. Sde proteins appeared critical for blocking access of membrane-disruptive early endosomal membrane material to the vacuole, as RNAi depletion of endosomal pathway components partially restored LCV integrity. The role of Sde proteins in preventing host degradation of the LCV was limited to the earliest stages of infection. The time that Sde proteins could prevent vacuole disruption, however, was extended by deletion of sidJ, which encodes a translocated protein that inactivates Sde protein active sites. These results indicate that Sde proteins act as temporally regulated vacuole guards during the establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments during the earliest steps of LCV biogenesis.

Identification of Genes Required for Long-Term Survival of Legionella pneumophila in Water.

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted l,d-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the wild type, as predicted for loss of an l,d-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions. IMPORTANCE Water is the primary vector for transmission of L. pneumophila to humans, and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires' disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42°C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan cross-linking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water.

The Sde Phosphoribosyl-Linked Ubiquitin Transferases protect the Legionella pneumophila vacuole from degradation by the host.

Legionella pneumophila grows intracellularly within a host membrane-bound vacuole that is formed in response to a bacterial type IV secretion system (T4SS). T4SS translocated Sde proteins promote phosphoribosyl-linked ubiquitination of endoplasmic reticulum protein Rtn4, but the role played by this modification is obscure due to lack of clear growth defects of mutants. To identify the steps in vacuole biogenesis promoted by these proteins, mutations were identified that unmasked growth defects in Δ sde strains. Mutations in the sdhA , ridL and legA3 genes aggravated the Δ sde fitness defect, resulting in disruption of the Legionella -containing vacuole (LCV) membrane within 2 hrs of bacterial contact with host cells. Depletion of Rab5B and sorting nexin 1 partially bypassed loss of Sde proteins, consistent with Sde blocking early endosome and retrograde trafficking, similar to roles previously demonstrated for SdhA and RidL proteins. Sde protein protection of LCV lysis was only observed shortly after infection, presumably because Sde proteins are inactivated by the metaeffector SidJ during the course of infection. Deletion of SidJ extended the time that Sde proteins could prevent vacuole disruption, indicating that Sde proteins are negatively regulated at the posttranslational level and are limited to protecting membrane integrity at the earliest stages of replication. Transcriptional analysis was consistent with this timing model for an early point of execution of Sde protein. Therefore, Sde proteins act as temporally-regulated vacuole guards during establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments early during biogenesis of the LCV. Significance statement: Maintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways, Legionella pneumophila Sde proteins that promote phosphoribosyl-linked ubiquitination of target eukaryotic proteins are shown to be temporally-regulated vacuole guards, preventing replication vacuole dissolution during early stages of infection. As targeting of reticulon 4 by these proteins leads to tubular endoplasmic reticulum aggregation, Sde proteins are likely to construct a barrier that blocks access of disruptive early endosomal compartments to the replication vacuole. Our study provides a new framework for how vacuole guards function to support biogenesis of the L. pneumophila replicative niche.

A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape.

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. ß-lactam targeting of penicillin-binding protein (PBP)-mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA-PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA-PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to ß-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various ß- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-ß-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore ß-lactam potency against gram-negative bacteria.

5' Untranslated mRNA Regions Allow Bypass of Host Cell Translation Inhibition by Legionella pneumophila.

Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a 2-h time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low-abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling data sets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5' untranslated regions (5' UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5' UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

Immunosuppression broadens evolutionary pathways to drug resistance and treatment failure during Acinetobacter baumannii pneumonia in mice.

Acinetobacter baumannii is increasingly refractory to antibiotic treatment in healthcare settings. As is true of most human pathogens, the genetic path to antimicrobial resistance (AMR) and the role that the immune system plays in modulating AMR during disease are poorly understood. Here we reproduced several routes to fluoroquinolone resistance, performing evolution experiments using sequential lung infections in mice that are replete with or depleted of neutrophils, providing two key insights into the evolution of drug resistance. First, neutropenic hosts acted as reservoirs for the accumulation of drug resistance during drug treatment. Selection for variants with altered drug sensitivity profiles arose readily in the absence of neutrophils, while immunocompetent animals restricted the appearance of these variants. Secondly, antibiotic treatment failure in the immunocompromised host was shown to occur without clinically defined resistance, an unexpected result that provides a model for how antibiotic failure occurs clinically in the absence of AMR. The genetic mechanism underlying both these results is initiated by mutations activating the drug egress pump regulator AdeL, which drives persistence in the presence of antibiotic. Therefore, antibiotic persistence mutations present a two-pronged risk during disease, causing drug treatment failure in the immunocompromised host while simultaneously increasing the emergence of high-level AMR.

SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase.

Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.

Yersinia pseudotuberculosis YopE prevents uptake by M cells and instigates M cell extrusion in human ileal enteroid-derived monolayers.

Many pathogens use M cells to access the underlying Peyer's patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb), and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyer's patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.

Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Previous work has posited that Sde proteins solely target serine (Ser) residues within acceptor protein substrates. We show here that SdeC-mediated pR-Ub modification results from a stepwise reaction that also modifies tyrosine (Tyr) residues. Unexpectedly, the presence of an HA tag on Ub resulted in poly-pR-ubiquitination, consistent with the HA tag acting as an acceptor target. Interrogation of phosphoribosyl-linked HA-Ub revealed that Tyr4 was the preferred targeted residue, based on LC-MS/MS analysis of the crosslinked product. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. Although previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr → Ser replacement mutants. This work demonstrates that pR-ubiquitination by the Sde family is not limited to Ser-modification as previously proposed, and broadens the potential sites targeted by this family.

Heightened Virulence of Yersinia Is Associated with Decreased Function of the YopJ Protein.

Despite the maintenance of YopP/J alleles throughout the human-pathogenic Yersinia lineage, the benefit of YopP/J-induced phagocyte death for Yersinia pathogenesis in animals is not obvious. To determine how the sequence divergence of YopP/J has impacted Yersinia virulence, we examined protein polymorphisms in this type III secreted effector protein across 17 Yersinia species and tested the consequences of polymorphism in a murine model of subacute systemic yersiniosis. Our evolutionary analysis revealed that codon 177 has been subjected to positive selection; the Yersinia enterocolitica residue had been altered from a leucine to a phenylalanine in nearly all Yersinia pseudotuberculosis and Yersinia pestis strains examined. Despite this change being minor, as both leucine and phenylalanine have hydrophobic side chains, reversion of YopJF177 to the ancestral YopJL177 variant yielded a Y. pseudotuberculosis strain with enhanced cytotoxicity toward macrophages, consistent with previous findings. Surprisingly, expression of YopJF177L in the mildly attenuated ksgA- background rendered the strain completely avirulent in mice. Consistent with this hypothesis that YopJ activity relates indirectly to Yersinia pathogenesis in vivo, ksgA- strains lacking functional YopJ failed to kill macrophages but actually regained virulence in animals. Also, treatment with the antiapoptosis drug suramin prevented YopJ-mediated macrophage cytotoxicity and enhanced Y. pseudotuberculosis virulence in vivo. Our results demonstrate that Yersinia-induced cell death is detrimental for bacterial pathogenesis in this animal model of illness and indicate that positive selection has driven YopJ/P and Yersinia evolution toward diminished cytotoxicity and increased virulence, respectively.

Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

Entropy of a bacterial stress response is a generalizable predictor for fitness and antibiotic sensitivity.

Current approaches explore bacterial genes that change transcriptionally upon stress exposure as diagnostics to predict antibiotic sensitivity. However, transcriptional changes are often specific to a species or antibiotic, limiting implementation to known settings only. While a generalizable approach, predicting bacterial fitness independent of strain, species or type of stress, would eliminate such limitations, it is unclear whether a stress-response can be universally captured. By generating a multi-stress and species RNA-Seq and experimental evolution dataset, we highlight the strengths and limitations of existing gene-panel based methods. Subsequently, we build a generalizable method around the observation that global transcriptional disorder seems to be a common, low-fitness, stress response. We quantify this disorder using entropy, which is a specific measure of randomness, and find that in low fitness cases increasing entropy and transcriptional disorder results from a loss of regulatory gene-dependencies. Using entropy as a single feature, we show that fitness and quantitative antibiotic sensitivity predictions can be made that generalize well beyond training data. Furthermore, we validate entropy-based predictions in 7 species under antibiotic and non-antibiotic conditions. By demonstrating the feasibility of universal predictions of bacterial fitness, this work establishes the fundamentals for potentially new approaches in infectious disease diagnostics.