Loss of the Escherichia coli inner membrane protein YhcB results in pleomorphic cell morphology and clear growth defects. Prior work suggested that YhcB was directly involved in cell division or peptidoglycan assembly. We found that loss of YhcB is detrimental in genetic backgrounds in which lipopolysaccharide (LPS) or glycerophospholipid (GPL) synthesis is altered. The growth defect of ΔyhcB could be rescued through inactivation of the Mla pathway, a system responsible for the retrograde transport of GPLs that are mislocalized to the outer leaflet of the outer membrane. Interestingly, this rescue was dependent upon the outer membrane phospholipase PldA that cleaves GPLs at the bacterial surface. Since the freed fatty acids resulting from PldA activity serve as a signal to the cell to increase LPS synthesis, this result suggested that outer membrane lipids are imbalanced in ΔyhcB. Mutations that arose in ΔyhcB populations during two independent suppressor screens were in genes encoding subunits of the acetyl coenzyme A carboxylase complex, which initiates fatty acid biosynthesis (FAB). These mutations fully restored cell morphology and reduced GPL levels, which were increased compared to wild-type bacteria. Growth of ΔyhcB with the FAB-targeting antibiotic cerulenin also increased cellular fitness. Furthermore, genetic manipulation of FAB and lipid biosynthesis showed that decreasing FAB rescued ΔyhcB filamentation, whereas increasing LPS alone could not. Altogether, these results suggest that YhcB may play a pivotal role in regulating FAB and, in turn, impact cell envelope assembly and cell division.IMPORTANCESynthesis of the Gram-negative cell envelope is a dynamic and complex process that entails careful coordination of many biosynthetic pathways. The inner and outer membranes are composed of molecules that are energy intensive to synthesize, and, accordingly, these synthetic pathways are under tight regulation. The robust nature of the Gram-negative outer membrane renders it naturally impermeable to many antibiotics and therefore a target of interest for antimicrobial design. Our data indicate that when the inner membrane protein YhcB is absent in Escherichia coli, the pathway for generating fatty acid substrates needed for all membrane lipid synthesis is dysregulated which leads to increased membrane material. These findings suggest a potentially novel regulatory mechanism for controlling the rate of fatty acid biosynthesis.

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). UPEC infects bladder epithelial cells (BECs) via fusiform vesicles and escapes into the cytosol by disrupting fusiform vesicle membrane using outer membrane phospholipase PldA, and establishes biofilm-like intracellular bacterial communities (IBCs) for protection from host immune clearance. Cytosolic UPEC is captured by autophagy to form autophagosomes, then transport to lysosomes, triggering the spontaneous exocytosis of lysosomes. The mechanism by which UPEC evades autophagy to recognize and form IBCs remains unclear. Here, we demonstrate that by inhibiting autophagic flux, UPEC PldA reduces the lysosome exocytosis of BECs. By reducing intracellular PI3P levels, UPEC PldA increases the accumulation of NDP52 granules and decreases the targeting of NDP52 to autophagy, hence stalling pre-autophagosome structures. Thus, our results uncover a critical role for PldA to inhibit autophagic flux, favoring UPEC escapes from lysosome exocytosis, thereby contributing to acute UTI.

Enteric bacteria need to adapt to endure the antibacterial activities of bile salts in the gut. Phospholipase A (PldA) is a key enzyme in the maintenance of bacterial membrane homeostasis. Bacteria respond to stress by modulating their membrane composition. Campylobacter jejuni is the most common cause of human worldwide. However, the mechanism by which C. jejuni adapts and survives in the gut environment is not fully understood. In this study, we investigated the roles of PldA, bile salt sodium deoxycholate (DOC), and oxygen availability in C. jejuni biology, mimicking an in vivo situation. Growth curves were used to determine the adaptation of C. jejuni to bile salts. RNA-seq and functional assays were employed to investigate the PldA-dependent and DOC-induced changes in gene expression that influence bacterial physiology. Survival studies were performed to address oxidative stress defense in C. jejuni. Here, we discovered that PldA of C. jejuni is required for optimal growth in the presence of bile salt DOC. Under high oxygen conditions, DOC is toxic to C. jejuni, but under low oxygen conditions, as is present in the lumen of the gut, C. jejuni benefits from DOC. C. jejuni PldA seems to enable the use of iron needed for optimal growth in the presence of DOC but makes the bacterium more vulnerable to oxidative stress. In conclusion, DOC stimulates C. jejuni growth under low oxygen conditions and alters colony morphology in a PldA-dependent manner. C. jejuni benefits from DOC by upregulating iron metabolism in a PldA-dependent manner.

Tetralogy of Fallot (TOF) is a cyanotic congenital condition contributed by genetic, epigenetic as well as environmental factors. We applied sparse machine learning algorithms to RNAseq and sRNAseq data to select the prospective biomarker candidates. Furthermore, we applied filtering techniques to identify a subset of biomarker pairs in TOF. Differential expression analysis disclosed 2757 genes and 214 miRNAs, which are dysregulated. Weighted gene co-expression network analysis on the differentially expressed genes extracted five significant modules that are enriched in GO terms, extracellular matrix, signaling and calcium ion binding. Also, voomNSC selected two genes and five miRNAs and transformed PLDA-predicted 72 genes and 38 miRNAs as prognostic biomarkers. Out of the selected biomarkers, miRNA target analysis revealed 14 miRNA-gene interactions. Also, 10 out of 14 pairs were oppositely expressed and four out of 10 oppositely expressed biomarker pairs shared common pathways of focal adhesion and P13K-Akt signaling. In conclusion, our study demonstrated the concept of biomarker pairs, which may be considered for clinical validation due to the high literature as well as experimental support.

Maintenance of phospholipid (PL) and lipopoly- or lipooligosaccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37°C). Loss of this protein had pleiotropic effects. A Y. pestis Δail mutant and KIM6+ wild type were systematically compared for (i) growth requirements at 37°C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernatants, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild-type phenotype. The Δail mutant grew normally at 28°C but lysed at 37°C when it entered stationary phase, as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immunofluorescence microscopy showed that the Δail mutant did not assemble Caf1 capsule. Expression of heat shock promoter rpoE or rpoH fused to a lux operon reporter were not induced when the Δail mutant was shifted from 28°C to 37°C (P < 0.001 and P < 0.01, respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca2+, or 2.5 mM Mg2+ to the growth medium or by a mutation in the phospholipase A gene (pldA::miniTn5, ΔpldA, or PldAS164A). A model accounting for the temperature-sensitive lysis of the Δail mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented. IMPORTANCE The Gram-negative pathogen Yersinia pestis transitions between a flea vector (ambient temperature) and a mammalian host (37°C). In response to 37°C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of ail destabilizes the OM at 37°C, causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.

Speaker recognition is a biometric modality that uses underlying speech information to determine the identity of the speaker. Speaker Identification (SID) under noisy conditions is one of the challenging topics in the field of speech processing, specifically when it comes to individuals with cochlear implants (CI). This study analyzes and quantifies the ability of CI-users to perform speaker identification based on direct electric auditory stimuli. CI users employ a limited number of frequency bands (8 ∼ 22) and use electrodes to directly stimulate the Basilar Membrane/Cochlear in order to recognize the speech signal. The sparsity of electric stimulation within the CI frequency range is a prime reason for loss in human speech recognition, as well as SID performance. Therefore, it is assumed that CI-users might be unable to recognize and distinguish a speaker given dependent information such as formant frequencies, pitch etc. which are lost to un-simulated electrodes. To quantify this assumption, the input speech signal is processed using a CI Advanced Combined Encoder (ACE) signal processing strategy to construct the CI auditory electrodogram. The proposed study uses 50 speakers from each of three different databases for training the system using two different classifiers under quiet, and tested under both quiet and noisy conditions. The objective result shows that, the CI users can effectively identify a limited number of speakers. However, their performance decreases when more speakers are added in the system, as well as when noisy conditions are introduced. This information could therefore be used for improving CI-user signal processing techniques to improve human SID.

Next-generation sequencing has emerged as an essential technology for the quantitative analysis of gene expression. In medical research, RNA sequencing (RNA-seq) data are commonly used to identify which type of disease a patient has. Because of the discrete nature of RNA-seq data, the existing statistical methods that have been developed for microarray data cannot be directly applied to RNA-seq data. Existing statistical methods usually model RNA-seq data by a discrete distribution, such as the Poisson, the negative binomial, or the mixture distribution with a point mass at zero and a Poisson distribution to further allow for data with an excess of zeros. Consequently, analytic tools corresponding to the above three discrete distributions have been developed: Poisson linear discriminant analysis (PLDA), negative binomial linear discriminant analysis (NBLDA), and zero-inflated Poisson logistic discriminant analysis (ZIPLDA). However, it is unclear what the real distributions would be for these classifications when applied to a new and real dataset. Considering that count datasets are frequently characterized by excess zeros and overdispersion, this paper extends the existing distribution to a mixture distribution with a point mass at zero and a negative binomial distribution and proposes a zero-inflated negative binomial logistic discriminant analysis (ZINBLDA) for classification. More importantly, we compare the above four classification methods from the perspective of model parameters, as an understanding of parameters is necessary for selecting the optimal method for RNA-seq data. Furthermore, we determine that the above four methods could transform into each other in some cases. Using simulation studies, we compare and evaluate the performance of these classification methods in a wide range of settings, and we also present a decision tree model created to help us select the optimal classifier for a new RNA-seq dataset. The results of the two real datasets coincide with the theory and simulation analysis results. The methods used in this work are implemented in the open-scource R scripts, with a source code freely available at https://github.com/FocusPaka/ZINBLDA.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.

Pseudomonas aeruginosa can manipulate eukaryotic host cells using secreted effectors delivered by the type III or the type VI Secretion Systems (T3SS and T6SS). The T3SS allows the injection of bacterial effectors (Exo toxins) into eukaryotic cell. P. aeruginosa, encodes three T6SSs, H1-, H2- and H3-T6SS. The H1-T6SS is mainly involved in delivering toxins to kill bacterial competitors. Recently, two T6SS-secreted phospholipases D, PldA (H2-T6SS) and PldB (H3-T6SS), were identified as trans-kingdom virulence effectors, triggering both killing of bacterial competitors and internalization into non-phagocytic cells. We deciphered the prevalence of T3SS and T6SS effectors encoding genes in 185 clinical isolates responsible for infections (septicaemia, pulmonary infections, urinary tract infections, and chronic infections in CF patients), 47 environmental strains, and on 33 carbapenemase-producers. We included 107 complete genomes of P. aeruginosa available in public databases. The prevalence of pldA is increased in clinical isolates responsible for severe acute infection and particularly in multi-drug resistant strains. In contrast, the pldB prevalence was high (96.8%) in all isolates. Regarding T3SS effectors, exoT and exoY are present in nearly all isolates while exoS and exoU were found to be exclusive with a higher prevalence of exoU + strains in severe acute infections. The hypervirulent exoU + isolates are more prone to be pldA +, suggesting a role of PldA in virulence. Finally, we observed that extremely drug resistant isolates producing an IMP-type carbapenemase were all pldA +. Our results suggest that PldA might have a role during pulmonary infections and have been co-selected in multidrug resistant strains particularly IMP-producers.

The outer membrane of Gram-negative bacteria is a critical barrier that prevents entry of noxious compounds. Integral to this functionality is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS), a molecule that is located exclusively in the outer leaflet of the outer membrane. Its lipid anchor, lipid A, is a glycolipid whose hydrophobicity and net negative charge are primarily responsible for the robustness of the membrane. Because of this, lipid A is a hallmark of Gram-negative physiology and is generally essential for survival. Rare exceptions have been described, including Acinetobacter baumannii, which can survive in the absence of lipid A, albeit with significant growth and membrane permeability defects. Here, we show by an evolution experiment that LOS-deficient A. baumannii can rapidly improve fitness over the course of only 120 generations. We identified two factors which negatively contribute to fitness in the absence of LOS, Mla and PldA. These proteins are involved in glycerophospholipid transport (Mla) and lipid degradation (PldA); both are active only on mislocalized, surface-exposed glycerophospholipids. Elimination of these two mechanisms was sufficient to cause a drastic fitness improvement in LOS-deficient A. baumannii The LOS-deficient double mutant grows as robustly as LOS-positive wild-type bacteria while remaining resistant to the last-resort polymyxin antibiotics. These data provide strong biological evidence for the directionality of Mla-mediated glycerophospholipid transport in Gram-negative bacteria and furthers our knowledge of asymmetry-maintenance mechanisms in the context of the outer membrane barrier.