Pathogenicity and virulence of Shigella sonnei: A highly drug-resistant pathogen of increasing prevalence.

Shigella spp. are the causative agent of shigellosis (or bacillary dysentery), a diarrhoeal disease characterized for the bacterial invasion of gut epithelial cells. Among the 4 species included in the genus, Shigella flexneri is principally responsible for the disease in the developing world while Shigella sonnei is the main causative agent in high-income countries. Remarkably, as more countries improve their socioeconomic conditions, we observe an increase in the relative prevalence of S. sonnei. To date, the reasons behind this change in aetiology depending on economic growth are not understood. S. flexneri has been widely used as a model to study the pathogenesis of the genus, but as more research data are collected, important discrepancies with S. sonnei have come to light. In comparison to S. flexneri, S. sonnei can be differentiated in numerous aspects; it presents a characteristic O-antigen identical to that of one serogroup of the environmental bacterium Plesiomonas shigelloides, a group 4 capsule, antibacterial mechanisms to outcompete and displace gut commensal bacteria, and a poorer adaptation to an intracellular lifestyle. In addition, the World Health Organization (WHO) have recognized the significant threat posed by antibiotic-resistant strains of S. sonnei, demanding new approaches. This review gathers knowledge on what is known about S. sonnei within the context of other Shigella spp. and aims to open the door for future research on understanding the increasing spread of this pathogen.

Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure.

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae, modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition. IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as "O antigen." This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae.

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.

The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells. TAKE AWAY: EspO homologues are found in EHEC, Shigella, S. typhimurium and some EPEC. EspO homologues interact with HAX-1. EspO protects infected cells from apoptosis. EspO joins a growing list of T3SS effectors that manipulate cell death pathways.

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen-host interactions.

Enteric pathogen-host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane-bound toll-like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro-inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1 and are regulated by related caspases, such as caspase-11, -4, -5 and -8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.

Mammalian lectin arrays for screening host-microbe interactions.

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen-receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.

Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence.

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation.

Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.

OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo.

Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost.

Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces.

Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing lesion on the surface of infected cells causes significant remodeling of the host cell surface; however, limited information is available about changes at the protein level. Here we employed plasma membrane profiling, a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor cluster of differentiation 55 (CD55) exhibited the greatest reduction in cell-surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.

Modulation of Host Cell Processes by T3SS Effectors.

Two of the enteric Escherichia coli pathotypes-enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)-have a conserved type 3 secretion system which is essential for virulence. The T3SS is used to translocate between 25 and 50 bacterial proteins directly into the host cytosol where they manipulate a variety of host cell processes to establish a successful infection. In this chapter, we discuss effectors from EPEC/EHEC in the context of the host proteins and processes that they target-the actin cytoskeleton, small guanosine triphosphatases and innate immune signalling pathways that regulate inflammation and cell death. Many of these translocated proteins have been extensively characterised, which has helped obtain insights into the mechanisms of pathogenesis of these bacteria and also understand the host pathways they target in more detail. With increasing knowledge of the positive and negative regulation of host signalling pathways by different effectors, a future challenge is to investigate how the specific effector repertoire of each strain cooperates over the course of an infection.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.

Shigella sonnei Does Not Use Amoebae as Protective Hosts.

Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered.IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei, as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections.

Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens.

Attaching and effacing (AE) pathogens colonize the gut mucosa using a type three secretion system (T3SS) and a suite of effector proteins. The locus of enterocyte effacement (LEE) is the defining genetic feature of the AE pathogens, encoding the T3SS and the core effector proteins necessary for pathogenesis. Extensive research has revealed a complex regulatory network that senses and responds to a myriad of host- and microbiota-derived signals in the infected gut to control transcription of the LEE. These signals include microbiota-liberated sugars and metabolites in the gut lumen, molecular oxygen at the gut epithelium, and host hormones. Recent research has revealed that AE pathogens also recognize physical signals, such as attachment to the epithelium, and that the act of effector translocation remodels gene expression in infecting bacteria. In this review, we summarize our knowledge to date and present an integrated view of how chemical, geographical, and physical cues regulate the virulence program of AE pathogens during infection.

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.

The pUltra plasmid series: A robust and flexible tool for fluorescent labeling of Enterobacteria.

Fluorescent labeling has been an invaluable tool for the study of living organisms and bacterial species are no exception to this. Here we present and characterize the pUltra plasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP) from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad range of Enterobacteria. The amount of expressed fluorophore from these genetic constructs is such, that the contours of the cells can be delineated on the basis of the fluorescent signal only. In addition, labeling through the pUltra plasmids can be used successfully for fluorescence and confocal microscopy while unambiguous distinction of cells labeled with different colors can be carried out efficiently by microscopy or flow cytometry. We compare the labeling provided by the pUltra plasmids with that of another plasmid series encoding fluorescent proteins and we show that the pUltra constructs are vastly superior in signal intensity and discrimination power without having any detectable growth rate effects for the bacterial population. We also use the pUltra plasmids to produce mixtures of differentially labeled pathogenic Escherichia, Shigella and Salmonella species which we test during infection of mammalian cells. We find that even inside the host cell, different strains can be distinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltra plasmids are a powerful labeling tool especially useful for complex biological experiments such as the visualization of ecosystems of different bacterial species or of enteric pathogens in contact with their hosts.

Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signaling axis to prevent recycling endosome maturation during infection.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.

Direct detection of lipid A on intact Gram-negative bacteria by MALDI-TOF mass spectrometry.

The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.