Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

The inner membrane protein YhiM links copper and CpxAR envelope stress responses in uropathogenic E. coli.

Urinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn5) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM, which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA, encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP. Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress.

Functional diversification of gram-negative intermembrane phospholipid transporters by intrinsic substrate preference.

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) of a strain with deletion of yhdP, but not tamB or ydbH, and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production. Deletion of tamB, redirecting phospholipid transport to YdbH, suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is directly related to phospholipid transport. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. A parsimonious explanation for our data is that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions.

Tracking DNA-based antigen-specific T cell receptors during progression to type 1 diabetes.

T cells targeting self-proteins are important mediators in autoimmune diseases. T cells express unique cell-surface receptors (TCRs) that recognize peptides presented by major histocompatibility molecules. TCRs have been identified from blood and pancreatic islets of individuals with type 1 diabetes (T1D). Here, we tracked ~1700 known antigen-specific TCR sequences, islet antigen or viral reactive, in bulk TCRß sequencing from longitudinal blood DNA samples in at-risk cases who progressed to T1D, age/sex/human leukocyte antigen-matched controls, and a new-onset T1D cohort. Shared and frequent antigen-specific TCRß sequences were identified in all three cohorts, and viral sequences were present across all ages. Islet sequences had different patterns of accumulation based upon antigen specificity in the at-risk cases. Furthermore, 73 islet-antigen TCRß sequences were present in higher frequencies and numbers in T1D samples relative to controls. The total number of these disease-associated TCRß sequences inversely correlated with age at clinical diagnosis, indicating the potential to use disease-relevant TCR sequences as biomarkers in autoimmune disorders.

Phosphatidylglycerol Is the Lipid Donor for Synthesis of Phospholipid-Linked Enterobacterial Common Antigen.

The Gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. In Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella, and Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPG contributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, in Escherichia coli K-12, the reaction synthesizing ECAPG from ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased the synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased the synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPG and provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor. IMPORTANCE The Gram-negative outer membrane is a permeability barrier preventing cellular entry of antibiotics. However, outer membrane biogenesis pathways are targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane of Enterobacterales (e.g., Escherichia, Salmonella, and Klebsiella). ECAPG consists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate-ECA is a phospholipid headgroup. The details of the reaction forming this molecule from polymerized ECA precursor are unknown. We determined the lipid donor providing the phospholipid moiety is phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPG provides the opportunity for development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat Gram-negative infections.

Temporal development of T cell receptor repertoires during childhood in health and disease.

T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next-generation sequencing technologies. However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. Here, we deep sequenced the TCR-ß chain repertoires from longitudinal peripheral blood DNA samples at 4 time points beginning early in life (median age of 1.4 years) from children who progressed to T1D (n = 29) and age/sex-matched islet autoantibody-negative controls (n = 25). From 53 million TCR-ß sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independently of disease. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and, separately, those specific for insulin and its precursor, preproinsulin. Insulin-reactive TCR-ß sequences were more common and frequent in number as the disease progressed in those who developed T1D compared with genetically at risk nondiabetic children, and this was not the case for influenza-reactive sequences. As an independent validation, we sequenced and analyzed TCR-ß repertoires from a cohort of new-onset T1D patients (n = 143), identifying the same preproinsulin-reactive TCRs. These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders.

Self-Antigens Targeted by Regulatory T Cells in Type 1 Diabetes.

While progress has been made toward understanding mechanisms that lead to the development of autoimmunity, there is less knowledge regarding protective mechanisms from developing such diseases. For example, in type 1 diabetes (T1D), the immune-mediated form of diabetes, the role of pathogenic T cells in the destruction of pancreatic islets is well characterized, but immune-mediated mechanisms that contribute to T1D protection have not been fully elucidated. One potential protective mechanism includes the suppression of immune responses by regulatory CD4 T cells (Tregs) that recognize self-peptides from islets presented by human leukocyte antigen (HLA) class II molecules. In this review, we summarize what is known about the antigenic self-peptides recognized by Tregs in the context of T1D.

Suppressor Mutants: History and Today's Applications.

For decades, biologist have exploited the near boundless advantages that molecular and genetic tools and analysis provide for our ability to understand biological systems. One of these genetic tools, suppressor analysis, has proven invaluable in furthering our understanding of biological processes and pathways and in discovering unknown interactions between genes and gene products. The power of suppressor analysis lies in its ability to discover genetic interactions in an unbiased manner, often leading to surprising discoveries. With advancements in technology, high-throughput approaches have aided in large-scale identification of suppressors and have helped provide insight into the core functional mechanisms through which suppressors act. In this review, we examine some of the fundamental discoveries that have been made possible through analysis of suppressor mutations. In addition, we cover the different types of suppressor mutants that can be isolated and the biological insights afforded by each type. Moreover, we provide considerations for the design of experiments to isolate suppressor mutants and for strategies to identify intergenic suppressor mutations. Finally, we provide guidance and example protocols for the isolation and mapping of suppressor mutants.

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen.

The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECAPG and lipopolysaccharide-linked ECALPS) and one periplasmic form of ECA (cyclic ECACYC). ECAPG, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECAPG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECAPG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECAPG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECAPG without other ECA forms, suggesting ECAPG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECAPG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECACYC. Our data demonstrate ECACYC can regulate ECAPG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECACYC act in a novel pathway that regulates the production of ECAPG, supporting a model in which ElyC provides feedback regulation of ECAPG production based on the periplasmic levels of ECACYC. IMPORTANCE Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales, including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECAPG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECACYC, genetically interact to inhibit the synthesis of ECAPG, potentially through feedback regulation based on ECACYC levels. This is the first insight into the pathway responsible for synthesis of ECAPG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECAPG levels.

CD4+ T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope.

Löfgren's syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3-restricted manner. Using ELISPOT analysis, a greater number of IFN-γ- and IL-2-secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

T-cell responses to hybrid insulin peptides prior to type 1 diabetes development.

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell-mediated destruction of insulin-producing ß-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

T cell receptor sequencing in autoimmunity.

T cells are an integral component of the adaptive immune response via the recognition of peptides by the cell surface-expressed T cell receptor (TCR). Rearrangement of the TCR genes results in a highly polymorphic repertoire on the T cells within a given individual. Although the diverse repertoire is beneficial for immune responses to foreign pathogens, recognition of self-peptides by T cells can contribute to the development of autoimmune disorders. Increasing evidence supports a pathogenic role for T cells in autoimmune pathology, and it is of interest to determine the TCR repertoires involved in autoimmune disease development. In this review, we summarize methodologies and advancements in the TCR sequencing field and discuss recent studies focused on TCR sequencing in a variety of autoimmune conditions. The rapidly evolving methodology of TCR sequencing has the potential to allow for a better understanding of autoimmune disease pathogenesis, identify disease-specific biomarkers, and aid in developing therapies to prevent and treat a number of these disorders.

The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli.

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and ß-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ∆yhdP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.

Enterobacterial Common Antigen: Synthesis and Function of an Enigmatic Molecule.

The outer membrane (OM) of Gram-negative bacteria poses a barrier to antibiotic entry due to its high impermeability. Thus, there is an urgent need to study the function and biogenesis of the OM. In Enterobacterales, an order of bacteria with many pathogenic members, one of the components of the OM is enterobacterial common antigen (ECA). We have known of the presence of ECA on the cell surface of Enterobacterales for many years, but its properties have only more recently begun to be unraveled. ECA is a carbohydrate antigen built of repeating units of three amino sugars, the structure of which is conserved throughout Enterobacterales. There are three forms of ECA, two of which (ECAPG and ECALPS) are located on the cell surface, while one (ECACYC) is located in the periplasm. Awareness of the importance of ECA has increased due to studies of its function that show it plays a vital role in bacterial physiology and interaction with the environment. Here, we review the discovery of ECA, the pathways for the biosynthesis of ECA, and the interactions of its various forms. In addition, we consider the role of ECA in the host immune response, as well as its potential roles in host-pathogen interaction. Furthermore, we explore recent work that offers insights into the cellular function of ECA. This review provides a glimpse of the biological significance of this enigmatic molecule.

Failed Genetic Protection: Type 1 Diabetes in the Presence of HLA-DQB1*06:02.

Certain HLA class II genes increase the risk for type 1 diabetes (T1D) development while others provide protection from disease development. HLA class II alleles encode MHC proteins on antigen-presenting cells, which function to present peptides and activate CD4 T cells. The DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype provides dominant protection across all stages of T1D and is a common haplotype found in Caucasians. However, it is present in <1% of people with T1D. Knowing which metabolic, immunologic, and genetic features are unique to individuals who fail genetic protection and develop T1D is important for defining the underlying mechanisms of DQB1*06:02-mediated protection. We describe a T1D cohort with DQB1*06:02 (n = 50) and compare them to individuals with T1D and without DQB1*06:02 (n = 2,759) who were identified over the last 26 years at the Barbara Davis Center for Diabetes. The age at diagnosis was similar between the cohorts and normally distributed throughout childhood and early adulthood. The average hemoglobin A1c was 10.8 ± 2.8% (95 ± 7 mmol/mol) at diagnosis in those DQB1*06:02 positive. The majority of T1D DQB1*06:02 + individuals were positive for one or more islet autoantibodies; however, there was a greater proportion who were islet autoantibody negative compared with those T1D DQB1*06:02 - individuals. Interestingly, DQB1*03:02, which confers significant T1D risk, was present in only those DQB1*06:02 + individuals with islet autoantibodies. This is one of the largest studies examining patients presenting with clinical T1D in the presence of DQB1*06:02, which provides a population to study the mechanisms of failed genetic protection against T1D.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Envelope stress responses: balancing damage repair and toxicity.

The Gram-negative envelope is a complex structure that consists of the inner membrane, the periplasm, peptidoglycan and the outer membrane, and protects the bacterial cell from the environment. Changing environmental conditions can cause damage, which triggers the envelope stress responses to maintain cellular homeostasis. In this Review, we explore the causes, both environmental and intrinsic, of envelope stress, as well as the cellular stress response pathways that counter these stresses. Furthermore, we discuss the damage to the cell that occurs when these pathways are aberrantly activated either in the absence of stress or to an excessive degree. Finally, we review the mechanisms whereby the σE response constantly acts to prevent cell death caused by highly toxic unfolded outer membrane proteins. Together, the recent work that we discuss has provided insights that emphasize the necessity for proper levels of stress response activation and the detrimental consequences that can occur in the absence of proper regulation.

A Stress Response Monitoring Lipoprotein Trafficking to the Outer Membrane.

Gram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles ß-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking. We used genetic and chemical biology approaches to induce several different lipoprotein trafficking stresses in Escherichia coli Our results identified the Cpx two-component system as a stress response for monitoring trafficking. Cpx is activated by trafficking defects and is required to protect the cell against the consequence of the resulting stress. The OM-targeted lipoprotein NlpE acts as a sensor that allows Cpx to gauge trafficking efficiency. We reveal that NlpE signals to Cpx while it is transiting the inner membrane (IM) en route to the OM and that only a small highly conserved N-terminal domain is required for signaling. We propose that defective trafficking causes NlpE to accumulate in the IM, activating Cpx to mount a transcriptional response that protects cells. Furthermore, we reconcile this new role of NlpE in signaling trafficking defects with its previously proposed role in sensing copper (Cu) stress by demonstrating that Cu impairs acylation of lipoproteins and, consequently, their trafficking to the OM.IMPORTANCE The outer membrane built by Gram-negative bacteria such as Escherichia coli forms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx help E. coli survive the assault of copper on a key outer membrane assembly pathway.

Genetic Analysis of Protein Translocation.

Cells in all domains of life must translocate newly synthesized proteins both across membranes and into membranes. In eukaryotes, proteins are translocated into the lumen of the ER or the ER membrane. In prokaryotes, proteins are translocated into the cytoplasmic membrane or through the membrane into the periplasm for Gram-negative bacteria or the extracellular space for Gram-positive bacteria. Much of what we know about protein translocation was learned through genetic selections and screens utilizing lacZ gene fusions in Escherichia coli. This review covers the basic principles of protein translocation and how they were discovered and developed. In particular, we discuss how lacZ gene fusions and the phenotypes conferred were exploited to identify the genes involved in protein translocation and provide insights into their mechanisms of action. These approaches, which allowed the elucidation of processes that are conserved throughout the domains of life, illustrate the power of seemingly simple experiments.

Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier of Escherichia coli in a Manner Controlled by YhdP.

Gram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.IMPORTANCE Enterobacterial common antigen (ECA) is a surface antigen made by all members of Enterobacteriaceae, including many clinically relevant genera (e.g., Escherichia, Klebsiella, Yersinia). Although this surface-exposed molecule is conserved throughout Enterobacteriaceae, very few functions have been ascribed to it. Here, we have determined that the periplasmic form of ECA, cyclic ECA, plays a role in maintaining the outer membrane permeability barrier. This activity is controlled by a protein of unknown function, YhdP, and deletion of yhdP damages the OM permeability barrier in a cyclic ECA-dependent manner, allowing harmful molecules such as antibiotics into the cell. This role in maintenance of the envelope permeability barrier is the first time a phenotype has been described for cyclic ECA. As the Gram-negative envelope is generally impermeable to antibiotics, understanding the mechanisms through which the barrier is maintained and antibiotics are excluded may lead to improved antibiotic delivery.