An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

Chemokine CX3CL1 (Fractalkine) Signaling and Diabetic Encephalopathy.

Diabetes mellitus (DM) is the most common metabolic disease in humans, and its prevalence is increasing worldwide in parallel with the obesity pandemic. A lack of insulin or insulin resistance, and consequently hyperglycemia, leads to many systemic disorders, among which diabetic encephalopathy (DE) is a long-term complication of the central nervous system (CNS), characterized by cognitive impairment and motor dysfunctions. The role of oxidative stress and neuroinflammation in the pathomechanism of DE has been proven. Fractalkine (CX3CL1) has unique properties as an adhesion molecule and chemoattractant, and by acting on its only receptor, CX3CR1, it regulates the activity of microglia in physiological states and neuroinflammation. Depending on the clinical context, CX3CL1-CX3CR1 signaling may have neuroprotective effects by inhibiting the inflammatory process in microglia or, conversely, maintaining/intensifying inflammation and neurotoxicity. This review discusses the evidence supporting that the CX3CL1-CX3CR1 pair is neuroprotective and other evidence that it is neurotoxic. Therefore, interrupting the vicious cycle within neuron-microglia interactions by promoting neuroprotective effects or inhibiting the neurotoxic effects of the CX3CL1-CX3CR1 signaling axis may be a therapeutic goal in DE by limiting the inflammatory response. However, the optimal approach to prevent DE is simply tight glycemic control, because the elimination of dysglycemic states in the CNS abolishes the fundamental mechanisms that induce this vicious cycle.

Anti-Inflammatory Action of Resveratrol in the Central Nervous System in Relation to Glucose Concentration-An In Vitro Study on a Blood-Brain Barrier Model.

Unbalanced blood glucose levels may cause inflammation within the central nervous system (CNS). This effect can be reversed by the action of a natural neuroprotective compound, resveratrol (RSV). The study aimed to investigate the anti-inflammatory effect of RSV on astrocyte cytokine profiles within an in vitro model of the blood-brain barrier (BBB) under varying glucose concentrations (2.2, 5.0, and 25.0 mmol/L), corresponding to hypo-, normo-, and hyperglycemia. The model included co-cultures of astrocytes (brain compartment, BC) and endothelial cells (microvascular compartment, MC), separated by 0.4 µm wide pores. Subsequent exposure to 0.2 µM LPS in the brain compartment (BC) and 50 µM RSV in the microvascular compartment (MC) of each well was carried out. Cytokine levels (IL-1 α, IL-1 ß, IL-2, IL-4, IL-6, IL-8) in the BC were assessed using a Multi-Analyte ELISArray Kit before and after the addition of LPS and RSV. Statistical analysis was performed to determine significance levels. The results demonstrated that RSV reduced the concentration of all studied cytokines in the BC, regardless of glucose levels, with the most substantial decrease observed under normoglycemic conditions. Additionally, the concentration of RSV in the BC was highest under normoglycemic conditions compared to hypo- and hyperglycemia. These findings confirm that administration of RSV in the MC exerts anti-inflammatory effects within the BC, particularly under normoglycemia-simulating conditions. Further in vivo studies, including animal and human research, are warranted to elucidate the bioavailability of RSV within the central nervous system (CNS).

The Role of Glucose Concentration and Resveratrol in Modulating Neuroinflammatory Cytokines: Insights from an In Vitro Blood-Brain Barrier Model.

BACKGROUND The prevalence of type 2 diabetes mellitus is rising, presumably because of a coexisting pandemic of obesity. Since diabetic neuropathy and neuroinflammation are frequent and significant complications of both prolonged hyperglycemia and iatrogenic hypoglycemia, the effect of glucose concentration and resveratrol (RSV) supplementation on cytokine profile was assessed in an in vitro model of the blood-brain barrier (BBB). MATERIAL AND METHODS The in vitro model of BBB was formed of endothelial cells and astrocytes, which represented the microvascular and brain compartments (MC and BC, respectively). The BC concentrations of selected cytokines - IL-10, IL-12, IL-17A, TNF-alpha, IFN-γ, GM-CSF in response to different glucose concentrations in the MC were studied. The influence of LPS in the BC and RSV in the MC on the cytokine profile in the BC was examined. RESULTS Low glucose concentration (40 mg/dL) in the MC resulted in increased concentration of all the cytokines in the BC except TNF-alpha, compared to normoglycemia-imitating conditions (90 mg/dL) (P<0.05). High glucose concentration (450 mg/dL) in the MC elevated the concentration of all the cytokines in the BC (P<0.05). RSV decreased the level of all cytokines in the BC after 24 h following its administration for all glucose concentrations in the MC (P<0.02). The greatest decline was observed in normoglycemic conditions (P<0.05). CONCLUSIONS Both hypo- and hyperglycemia-simulating conditions impair the cytokine profile in BC, while RSV can normalize it, despite relatively poor penetration through the BBB. RSV exhibits anti-neuroinflammatory effects, especially in the group with normoglycemia-simulating conditions.

Effects of Diabetes Mellitus-Related Dysglycemia on the Functions of Blood-Brain Barrier and the Risk of Dementia.

Diabetes mellitus is one of the most common metabolic diseases worldwide, and its long-term complications include neuropathy, referring both to the peripheral and to the central nervous system. Detrimental effects of dysglycemia, especially hyperglycemia, on the structure and function of the blood-brain barrier (BBB), seem to be a significant backgrounds of diabetic neuropathy pertaining to the central nervous system (CNS). Effects of hyperglycemia, including excessive glucose influx to insulin-independent cells, may induce oxidative stress and secondary innate immunity dependent inflammatory response, which can damage cells within the CNS, thus promoting neurodegeneration and dementia. Advanced glycation end products (AGE) may exert similar, pro-inflammatory effects through activating receptors for advanced glycation end products (RAGE), as well as some pattern-recognition receptors (PRR). Moreover, long-term hyperglycemia can promote brain insulin resistance, which may in turn promote Aß aggregate accumulation and tau hyperphosphorylation. This review is focused on a detailed analysis of the effects mentioned above towards the CNS, with special regard to mechanisms taking part in the pathogenesis of central long-term complications of diabetes mellitus initiated by the loss of BBB integrity.

Interplay between Systemic Glycemia and Neuroprotective Activity of Resveratrol in Modulating Astrocyte SIRT1 Response to Neuroinflammation.

The flow of substances between the blood and the central nervous system is precisely regulated by the blood-brain barrier (BBB). Its disruption due to unbalanced blood glucose levels (hyper- and hypoglycemia) occurring in metabolic disorders, such as type 2 diabetes, can lead to neuroinflammation, and increase the risk of developing neurodegenerative diseases. One of the most studied natural anti-diabetic, anti-inflammatory, and neuroprotective compounds is resveratrol (RSV). It activates sirtuin 1 (SIRT1), a key metabolism regulator dependent on cell energy status. The aim of this study was to assess the astrocyte SIRT1 response to neuroinflammation and subsequent RSV treatment, depending on systemic glycemia. For this purpose, we used an optimized in vitro model of the BBB consisting of endothelial cells and astrocytes, representing microvascular and brain compartments (MC and BC), in different glycemic backgrounds. Astrocyte-secreted SIRT1 reached the highest concentration in hypo-, the lowest in normo-, and the lowest in hyperglycemic backgrounds. Lipopolysaccharide (LPS)-induced neuroinflammation caused a substantial decrease in SIRT1 in all glycemic backgrounds, as observed earliest in hyperglycemia. RSV partially counterbalanced the effect of LPS on SIRT1 secretion, most remarkably in normoglycemia. Our results suggest that abnormal glycemic states have a worse prognosis for RSV-therapy effectiveness compared to normoglycemia.

Translational regulation in mycobacteria and its implications for pathogenicity.

Protein synthesis is a fundamental requirement of all cells for survival and replication. To date, vast numbers of genetic and biochemical studies have been performed to address the mechanisms of translation and its regulation in Escherichia coli, but only a limited number of studies have investigated these processes in other bacteria, particularly in slow growing bacteria like Mycobacterium tuberculosis, the causative agent of human tuberculosis. In this Review, we highlight important differences in the translational machinery of M. tuberculosis compared with E. coli, specifically the presence of two additional proteins and subunit stabilizing elements such as the B9 bridge. We also consider the role of leaderless translation in the ability of M. tuberculosis to establish latent infection and look at the experimental evidence that translational regulatory mechanisms operate in mycobacteria during stress adaptation, particularly focussing on differences in toxin-antitoxin systems between E. coli and M. tuberculosis and on the role of tuneable translational fidelity in conferring phenotypic antibiotic resistance. Finally, we consider the implications of these differences in the context of the biological adaptation of M. tuberculosis and discuss how these regulatory mechanisms could aid in the development of novel therapeutics for tuberculosis.

Functional genetic variation in pe/ppe genes contributes to diversity in Mycobacterium tuberculosis lineages and potential interactions with the human host.

Introduction: Around 10% of the coding potential of Mycobacterium tuberculosisis constituted by two poorly understood gene families, the pe and ppe loci, thought to be involved in host-pathogen interactions. Their repetitive nature and high GC content have hindered sequence analysis, leading to exclusion from whole-genome studies. Understanding the genetic diversity of pe/ppe families is essential to facilitate their potential translation into tools for tuberculosis prevention and treatment. Methods: To investigate the genetic diversity of the 169 pe/ppe genes, we performed a sequence analysis across 73 long-read assemblies representing seven different lineages of M. tuberculosis and M. bovis BCG. Individual pe/ppe gene alignments were extracted and diversity and conservation across the different lineages studied. Results: The pe/ppe genes were classified into three groups based on the level of protein sequence conservation relative to H37Rv, finding that >50% were conserved, with indels in pe_pgrs and ppe_mptr sub-families being major drivers of structural variation. Gene rearrangements, such as duplications and gene fusions, were observed between pe and pe_pgrs genes. Inter-lineage diversity revealed lineage-specific SNPs and indels. Discussion: The high level of pe/ppe genes conservation, together with the lineage-specific findings, suggest their phylogenetic informativeness. However, structural variants and gene rearrangements differing from the reference were also identified, with potential implications for pathogenicity. Overall, improving our knowledge of these complex gene families may have insights into pathogenicity and inform the development of much-needed tools for tuberculosis control.

Association analysis between symptomology and herpesvirus IgG antibody concentrations in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis (MS) are two complex and multifactorial diseases whose patients experience persistent fatigue, cognitive impairment, among other shared symptoms. The onset of these diseases has also been linked to acute herpesvirus infections or their reactivations. In this work, we re-analyzed a previously-described dataset related to IgG antibody responses to 6 herpesviruses (CMV - cytomegalovirus; EBV - Epstein-Barr virus; HHV6 - human herpesvirus-6; HSV1 and HSV2 - herpes simplex virus-1 and -2, respectively; VZV - varicella-zoster virus) from the United Kingdom ME/CFS biobank. The primary goal was to report the underlying symptomology and its association with herpesvirus IgG antibodies using data from 4 disease-trigger-based subgroups of ME/CFS patients (n = 222) and patients with MS (n = 46). The secondary objective was to assess whether serological data could distinguish ME/CFS and its subgroup from MS using a SuperLearner (SL) algorithm. There was evidence for a significant negative association between temporary eye insight disturbance and CMV antibody concentrations and for a significant positive association between bladder problems and EBV antibody concentrations in the MS group. In the ME/CFS or its subgroups, the most significant antibody-symptom association was obtained for increasing HSV1 antibody concentration and brain fog, a finding in line with a negative impact of HSV1 exposure on cognitive outcomes in both healthy and disease conditions. There was also evidence for a higher number of significant antibody-symptom associations in the MS group than in the ME/CFS group. When we combined all the serological data in an SL algorithm, we could distinguish three ME/CFS subgroups (unknown disease trigger, non-infection trigger, and an infection disease trigger confirmed in the lab at the time of the event) from the MS group. However, we could not find the same for the remaining ME/CFS group (related to an unconfirmed infection disease). In conclusion, IgG antibody data explains more the symptomology of MS patients than the one of ME/CFS patients. Given the fluctuating nature of symptoms in ME/CFS patients, the clinical implication of these findings remains to be determined with a longitudinal study. This study is likely to ascertain the robustness of the associations during natural disease course.

Revisiting IgG Antibody Reactivity to Epstein-Barr Virus in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Its Potential Application to Disease Diagnosis.

Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients.

Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism.

BACKGROUND: Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain. RESULTS: In the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter. CONCLUSIONS: The present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The dba expression is not only essential for the translation of the downstream dsbI gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI.

Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5' leader region, which usually contains the Shine-Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin-antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine-Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine-Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine-Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine-Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine-Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: analysis of high-throughput epigenetic and gene expression studies.

Patients affected by Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) show specific epigenetic and gene expression signatures of the disease. However, it is unknown whether these signatures include abnormal levels of the human angiotensin-converting enzymes, ACE and ACE2, the latter being the main receptor described for the host-cell invasion by SARS-CoV-2. To investigate that, we first re-analyzed available case-control epigenome-wide association studies based on DNA methylation data, and case-control gene expression studies based on microarray data. From these published studies, we found an association between ME/CFS and 4 potentially hypomethylated probes located in the ACE locus. We also found another disease association with one hypomethylated probe located in the transcription start site of ACE2. The same disease associations were obtained for women but not for men after performing sex-specific analyses. In contrast, a meta-analysis of gene expression levels could not provide evidence for a differentially expression of ACE and ACE2 in affected patients when compared to healthy controls. In line with this negative finding, the analysis of a new data set on the gene expression of ACE and ACE2 in peripheral blood mononuclear cells did not find any differences between a female cohort of 37 patients and 34 age-matched healthy controls. Future studies should be conducted to extend this investigation to other potential receptors used by SARS-CoV-2. These studies will help researchers and clinicians to improve the understanding of the health risk imposed by this virus when infecting patients affected by this debilitating disease.

The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) in myalgic encephalomyelitis/chronic fatigue syndrome: A meta-analysis of public DNA methylation and gene expression data.

People with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often report a high frequency of viral infections and flu-like symptoms during their disease course. Given that this reporting agrees with different immunological abnormalities and altered gene expression profiles observed in the disease, we aimed at answering whether the expression of the human angiotensin-converting enzyme 2 (ACE2), the major cell entry receptor for SARS-CoV-2, is also altered in these patients. In particular, a low expression of ACE2 could be indicative of a high risk of developing COVID-19. We then performed a meta-analysis of public data on CpG DNA methylation and gene expression of this enzyme and its homologous ACE protein in peripheral blood mononuclear cells and related subsets. We found that patients with ME/CFS have decreased methylation levels of four CpG probes in the ACE locus (cg09920557, cg19802564, cg21094739, and cg10468385) and of another probe in the promoter region of the ACE2 gene (cg08559914). We also found a decreased expression of ACE2 but not of ACE in patients when compared to healthy controls. Accordingly, in newly collected data, there was evidence for a significant higher proportion of samples with an ACE2 expression below the limit of detection in patients than healthy controls. Altogether, patients with ME/CFS can be at a higher COVID-19 risk and, if so, they should be considered a priority group for vaccination by public health authorities. To further support this conclusion, similar research is recommended for other human cell entry receptors and cell types, namely, those cells targeted by the virus.

Herpesviruses Serology Distinguishes Different Subgroups of Patients From the United Kingdom Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Biobank.

The evidence of an association between Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and chronic herpesviruses infections remains inconclusive. Two reasons for the lack of consistent evidence are the large heterogeneity of the patients' population with different disease triggers and the use of arbitrary cutoffs for defining seropositivity. In this work we re-analyzed previously published serological data related to 7 herpesvirus antigens. Patients with ME/CFS were subdivided into four subgroups related to the disease triggers: S0-42 patients who did not know their disease trigger; S1-43 patients who reported a non-infection trigger; S2-93 patients who reported an infection trigger, but that infection was not confirmed by a lab test; and S3-48 patients who reported an infection trigger and that infection was confirmed by a lab test. In accordance with a sensitivity analysis, the data were compared to those from 99 healthy controls allowing the seropositivity cutoffs to vary within a wide range of possible values. We found a negative association between S1 and seropositivity to Epstein-Barr virus (VCA and EBNA1 antigens) and Varicella-Zoster virus using specific seropositivity cutoff. However, this association was not significant when controlling for multiple testing. We also found that S3 had a lower seroprevalence to the human cytomegalovirus when compared to healthy controls for all cutoffs used for seropositivity and after adjusting for multiple testing using the Benjamini-Hochberg procedure. However, this association did not reach statistical significance when using Benjamini-Yekutieli procedure. In summary, herpesviruses serology could distinguish subgroups of ME/CFS patients according to their disease trigger, but this finding could be eventually affected by the problem of multiple testing.

Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System.

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases.

Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

Sodium Taurocholate Stimulates Campylobacter jejuni Outer Membrane Vesicle Production via Down-Regulation of the Maintenance of Lipid Asymmetry Pathway.

Campylobacter jejuni outer membrane vesicles (OMVs) contain numerous virulence-associated proteins including the cytolethal distending toxin and three serine proteases. As C. jejuni lacks the classical virulence-associated secretion systems of other enteric pathogens that deliver effectors directly into target cells, OMVs may have a particularly important role in virulence. C. jejuni OMV production is stimulated by the presence of physiological concentrations of the bile salt sodium taurocholate (ST) through an unknown mechanism. The maintenance of lipid asymmetry (MLA) pathway has been implicated in a novel mechanism for OMV biogenesis, open to regulation by host signals. In this study we investigated the role of the MLA pathway in C. jejuni OMV biogenesis with ST as a potential regulator. OMV production was quantified by analyzing protein and lipid concentrations of OMV preparations and OMV particle counts produced by nanoparticle tracking analysis. Mutation of mlaA which encodes the outer membrane component of the MLA pathway significantly increased OMV production compared to the wild-type strain. Detergent sensitivity and membrane permeability assays confirmed the increased OMV production was not due to changes in membrane stability. The presence of 0.2% (w/v) ST increased wild-type OMV production and reduced OMV size, but did not further stimulate mlaA mutant OMV production or significantly alter mlaA mutant OMV size. qRT-PCR analysis demonstrated that the presence of ST decreased expression of both mlaA and mlaC in C. jejuni wild-type strains 11168 and 488. Collectively the data in this study suggests C. jejuni can regulate OMV production in response to host gut signals through changes in expression of the MLA pathway. As the gut bile composition is dependent on both diet and the microbiota, this study highlights the potential importance of diet and lifestyle factors on the varying disease presentations associated with gut pathogen infection.