Gram-negative Type Research Articles

The type II secretion system as an underappreciated and understudied mediator of interbacterial antagonism.

Interbacterial antagonism involves all major phyla, occurs across the full range of ecological niches, and has great significance for the environment, clinical arena, and agricultural and industrial sectors. Though the earliest insight into interbacterial antagonism traces back to the discovery of antibiotics, a paradigm shift happened when it was learned that protein secretion systems (e.g., types VI and IV secretion systems) deliver toxic "effectors" against competitors. However, a link between interbacterial antagonism and the Gram-negative type II secretion system (T2SS), which exists in many pathogens and environmental species, is not evident in prior reviews on bacterial competition or T2SS function. A current examination of the literature revealed four examples of a T2SS or one of its known substrates having a bactericidal activity against a Gram-positive target or another Gram-negative. When further studied, the T2SS effectors proved to be peptidases that target the peptidoglycan of the competitor. There are also reports of various bacteriolytic enzymes occurring in the culture supernatants of some other Gram-negative species, and a link between these bactericidal activities and T2SS is suggested. Thus, a T2SS can be a mediator of interbacterial antagonism, and it is possible that many T2SSs have antibacterial outputs. Yet, at present, the T2SS remains relatively understudied for its role in interbacterial competition. Arguably, there is a need to analyze the T2SSs of a broader range of species for their role in interbacterial antagonism. Such investigation offers, among other things, a possible pathway toward developing new antimicrobials for treating disease.

A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae.

Gram-negative bacteria use type VI secretion system (T6SS) to deliver effectors that interact with neighboring cells for niche advantage. Klebsiella pneumoniae is an opportunistic nosocomial pathogen that often carries multiple T6SS loci, the function of which has not yet been elucidated. We performed a genomic analysis of 65 clinical K. pneumoniae strains isolated from various sources, confirming that all strains contained T6SS. We then used transcriptomics to further study changes in gene expression and its effect on interbacterial competition following the knockout of key T6SS genes in sequence type 11 (ST11) K. pneumoniae HS11286. Our findings revealed the distribution and genomic characteristics of T6SS in clinical K. pneumoniae. This study also described the overall transcriptional changes in the predominant Chinese ST11 strain HS11286 upon deletion of crucial T6SS genes. Additionally, this work provides a reference for future research on the identification of T6SS in bacteria.

Isolation of Multidrug-Resistant Bacteria as an Independent Mortality Factor in Patients with Suspected Sepsis at National Medical Center of the West

Introduction: In the changing epidemiological landscape sepsis corresponds to one of the main causes of in-hospital mortality, in third level care units the presence of multidrug resistant bacteria to antimicrobials correspond to a severe health problem, there are studies in intensive care units on mortality related to the appearance of multidrug resistant bacteria in cultures, however infection by multidrug resistant bacteria can appear at any instant of hospital stay which can become an independent risk factor for the appearance of mortality. Aims: To associate the isolation of multidrug-resistant bacteria as an independent mortality factor in patients with suspected sepsis at the Centro Médico Nacional De Occidente. Materials and methods: Place of study: Centro Médico Nacional De Occidente Guadalajara, Jalisco, Mexico. Type of study: Clinical, retrospective cohort type. The clinical records of patients admitted during the study period were searched. Patients with suspected sepsis according to the definition of the Third International Consensus were identified from continuous medical admission and followed up throughout their stay in the hospital. Patients with culture results were evaluated. Two follow-up cohorts were identified: those who presented isolation of multidrug-resistant bacteria at any time during hospitalization and the second cohort those who did not present isolation of multidrug-resistant bacteria; an initial determination of the variables was made and then the clinical records of the patients during their stay in the services were reviewed to search for in-hospital mortality. The association between the occurrence of multidrug-resistant bacteria and mortality was estimated by Hazard Ratio (with 95% confidence intervals). Results: A total of 94 patients were included with a minimum age of 16 years and a maximum age of 91 years with a mean of 48 years with a standard deviation of 20 years. A total of 93 cultures were included of which 21 (22.3%) were isolated with bacterial resistance. Of the positive cultures 42 % (9) were blood culture, 10 % (2) were surgical wound cultures, urine culture 23 % (5), bronchial aspirate 23 % (5). Regarding mortality, 57 % of the patients who presented bacterial resistance presented mortality, presenting a Hazard Ratio of 3.371 with 95% CI (1.125-10.100) p=0.02 Table 2 and 3. Regarding mortality with the different types of bacteria, it was 40 % in Gram negative bacteria with a Hazard Ratio of 1.875 (0.291-12.089), while it was 20 % in Gram positive bacteria, which was not statistically significant p=0.3. Conclusion: Isolation of multidrug-resistant bacteria is an independent mortality factor in patients with suspected sepsis at Centro Médico Nacional De Occidente. There is no relationship with the isolation of Gram-positive and Gram-negative bacteria type with respect to patient mortality.

Distribution and diversity of type VI secretion system clusters in Enterobacter bugandensis and Enterobacter cloacae.

Gram-negative bacteria use type VI secretion systems (T6SSs) to antagonize neighbouring cells. Although primarily involved in bacterial competition, the T6SS is also implicated in pathogenesis, biofilm formation and ion scavenging. Enterobacter species belong to the ESKAPE pathogens, and while their antibiotic resistance has been well studied, less is known about their pathogenesis. Here, we investigated the distribution and diversity of T6SS components in isolates of two clinically relevant Enterobacter species, E. cloacae and E. bugandensis. T6SS clusters are grouped into four types (T6SSi-T6SSiv), of which type i can be further divided into six subtypes (i1, i2, i3, i4a, i4b, i5). Analysis of a curated dataset of 31 strains demonstrated that most of them encode T6SS clusters belonging to the T6SSi type. All T6SS-positive strains possessed a conserved i3 cluster, and many harboured one or two additional i2 clusters. These clusters were less conserved, and some strains displayed evidence of deletion. We focused on a pathogenic E. bugandensis clinical isolate for comprehensive in silico effector prediction, with comparative analyses across the 31 isolates. Several new effector candidates were identified, including an evolved VgrG with a metallopeptidase domain and a Tse6-like protein. Additional effectors included an anti-eukaryotic catalase (KatN), M23 peptidase, PAAR and VgrG proteins. Our findings highlight the diversity of Enterobacter T6SSs and reveal new putative effectors that may be important for the interaction of these species with neighbouring cells and their environment.

Substrate recruitment mechanism by gram-negative type III, IV, and VI bacterial injectisomes.

Bacteria use a wide arsenal of macromolecular substrates (DNA and proteins) to interact with or infect prokaryotic and eukaryotic cells. To do so, they utilize substrate-injecting secretion systems or injectisomes. However, prior to secretion, substrates must be recruited to specialized recruitment platforms and then handed over to the secretion apparatus for secretion. In this review, we provide an update on recent advances in substrate recruitment and delivery by gram-negative bacterial recruitment platforms associated with Type III, IV, and VI secretion systems.

Paradoxical Activation of a Type VI Secretion System Phospholipase Effector by Its Cognate Immunity Protein.

Type VI secretion systems (T6SSs) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce autopermeabilization through unopposed activity of the Tle phospholipase effector. This hyperpermeability phenotype is T6SS dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyperpermeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyperpermeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG β-spike protein. Together, these findings indicate that Tli has distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export. IMPORTANCE Gram-negative bacteria use type VI secretion systems deliver toxic effector proteins directly into neighboring competitors. Secreting cells also produce specific immunity proteins that neutralize effector activities to prevent autointoxication. Here, we show the Tli immunity protein of Enterobacter cloacae has two distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to block Tle lipase effector activity, while cytoplasmic Tli is required to activate the lipase prior to export. These results indicate Tle interacts transiently with its cognate immunity protein to promote effector protein folding and/or packaging into the secretion apparatus.

Monitoring bromide loss in bromoacetyl-derivatized polyribosylribitol polysaccharide in Haemophilus influenzae type b for PedvaxHIB® by capillary electrophoresis and NMR spectroscopy

PedvaxHIB® is an effective pediatric vaccine for protecting infants from invasive gram-negative bacterium Haemophilus influenzae type b. It is a highly purified capsular polysaccharide, polyribosylribitol phosphate that is covalently linked to an outer membrane protein complex of Neisseria meningitidis. PRP is first derivatized with an organic linker, followed by the coupling of a butadiamine group, and then at the end terminal, a bromoacetyl group is attached for conjugation with thiolated OMPC. The stability of the bromide group in derivatized PRP is monitored by two different methods, capillary electrophoresis and NMR spectroscopy. The loss of the bromide group is detected by measuring the amount of free bromide ion liberated using capillary electrophoresis and by observing a change in amide proton peaks near the bromide group using NMR. The two methods give similar rate hydrolysis results, therefore both can be employed as quick stability tools for bromoacetylation PRP content during manufacturing.

Host manipulation by bacterial type III and type IV secretion system effector proteases.

Proteases are powerful enzymes, which cleave peptide bonds, leading most of the time to irreversible fragmentation or degradation of their substrates. Therefore they control many critical cell fate decisions in eukaryotes. Bacterial pathogens exploit this power and deliver protease effectors through specialised secretion systems into host cells. Research over the past years revealed that the functions of protease effectors during infection are diverse, reflecting the lifestyles and adaptations to specific hosts; however, only a small number of peptidase families seem to have given rise to most of these protease virulence factors by the evolution of different substrate-binding specificities, intracellular activation and subcellular targeting mechanisms. Here, we review our current knowledge about the enzymology and function of protease effectors, which Gram-negative bacterial pathogens translocate via type III and IV secretion systems to irreversibly manipulate host processes. We highlight emerging concepts such as signalling by protease cleavage products and effector-triggered immunity, which host cells employ to detect and defend themselves against a protease attack. TAKE AWAY: Proteases irreversibly cleave proteins to control critical cell fate decisions. Gram-negative bacteria use type III and IV secretion systems to inject effectors. Protease effectors are integral weapons for the manipulation of host processes. Effectors evolved from few peptidase families to target diverse substrates. Effector-triggered immunity upon proteolytic attack emerges as host defence.

IT4SE-EP: Accurate Identification of Bacterial Type IV Secreted Effectors by Exploring Evolutionary Features from Two PSI-BLAST Profiles.

Many gram-negative bacteria use type IV secretion systems to deliver effector molecules to a wide range of target cells. These substrate proteins, which are called type IV secreted effectors (T4SE), manipulate host cell processes during infection, often resulting in severe diseases or even death of the host. Therefore, identification of putative T4SEs has become a very active research topic in bioinformatics due to its vital roles in understanding host-pathogen interactions. PSI-BLAST profiles have been experimentally validated to provide important and discriminatory evolutionary information for various protein classification tasks. In the present study, an accurate computational predictor termed iT4SE-EP was developed for identifying T4SEs by extracting evolutionary features from the position-specific scoring matrix and the position-specific frequency matrix profiles. First, four types of encoding strategies were designed to transform protein sequences into fixed-length feature vectors based on the two profiles. Then, the feature selection technique based on the random forest algorithm was utilized to reduce redundant or irrelevant features without much loss of information. Finally, the optimal features were input into a support vector machine classifier to carry out the prediction of T4SEs. Our experimental results demonstrated that iT4SE-EP outperformed most of existing methods based on the independent dataset test.

Modification of zeolitic imidazolate framework-8 with amine groups for improved antibacterial activity

The exploration of the metal–organic framework (MOFs) as a promising alternative for disinfection applications has not yet widely recognized. The flexibility of its structural diversity and large surface area with large pore size allows guest molecules to be stored and their ability to release metal ions has provided great prospect to generate high potent antibacterial activity for environmental applications. In this study, a rapid and straightforward post modification method for zeolitic imidazole framework 8 (ZIF-8) was developed through amine groups' introduction using ammonium hydroxide solution with improved antibacterial properties. The effect of different volume of ammonium hydroxide loadings towards ZIF-8 properties and its antibacterial activity was carefully examined. The result of Fourier transform infrared spectroscopy (FTIR) showed a successful modification of ZIF-8 with amine due to the presence of the N–H group; however, a significant decrease was observed in the BET surface area. The samples' antibacterial potency was tested against Escherichia coli ATCC 11229 and Staphylococcus aureus ATCC 6538 as Gram-negative and positive bacteria type strains, using disc diffusion (DDT) and minimum inhibitory concentration (MIC) testing, respectively. ZNH 20 was found to have the most potent antibacterial efficacy, with the MIC value needed to prevent bacterial growth two times lower than that of unmodified ZIF-8. The presence of positively charged amine groups promotes electrostatic adsorption of negatively charged membrane bacteria cells, which substantially enhanced the antibacterial activity of the modified sample. The synergistic effects of amine groups as well as zinc metal (Zn2+) in the modified ZIF-8 increased the antibacterial activity of the material and thus, making it suitable as antibacterial agents for water purification and biomedical applications.

Impact of phosphate dosing on the microbial ecology of drinking water distribution systems: Fieldwork studies in chlorinated networks

Phosphate is routinely dosed to ensure regulatory compliance for lead in drinking water distribution systems. Little is known about the impact of the phosphate dose on the microbial ecology in these systems and in particular the endemic biofilms. Disturbance of the biofilms and embedded material in distribution can cause regulatory failures for turbidity and metals. To investigate the impact of phosphate on developing biofilms, pipe wall material from four independent pipe sections was mobilised and collected using two twin-flushing operations a year apart in a chlorinated UK network pre- and post-phosphate dosing. Intensive monitoring was undertaken, including turbidity and water physico-chemistry, traditional microbial culture-based indicators, and microbial community structure via sequencing the 16S rRNA gene for bacteria and the ITS2 gene for fungi. Whole metagenome sequencing was used to study shifts in functional characteristics following the addition of phosphate.As an operational consequence, turbidity responses from the phosphate-enriched water were increased, particularly from cast iron pipes. Differences in the taxonomic composition of both bacteria and fungi were also observed, emphasising a community shift towards microorganisms able to use or metabolise phosphate. Phosphate increased the relative abundance of bacteria such as Pseudomonas, Paenibacillus, Massilia, Acinetobacter and the fungi Cadophora, Rhizophagus and Eupenicillium. Whole metagenome sequencing showed with phosphate a favouring of sequences related to Gram-negative bacterium type cell wall function, virions and thylakoids, but a reduction in the number of sequences associated to vitamin binding, methanogenesis and toxin biosynthesis. With current faecal indicator tests only providing risk detection in bulk water samples, this work improves understanding of how network changes effect microbial ecology and highlights the potential for new approaches to inform future monitoring or control strategies to protect drinking water quality.

Are all faecal bacteria detected with equal efficiency? A study using next-generation sequencing and quantitative culture of infants' faecal samples

BackgroundMany species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota. MethodsTwenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1–V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains. ResultsNGS yielded one read per 106 CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 108 CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >106 CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria. ConclusionsNGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy.

High-yield production of recombinant platelet factor 4 by harnessing and honing the gram-negative bacterial secretory apparatus

Platelet factor 4 is a cytokine released into the bloodstream by activated platelets where it plays a pivotal role in etiology and diagnosis of heparin-induced thrombocytopenia. Therefore, a sustainable source of recombinant PF4 with structural and functional similarity to its native form is urgently needed to be used in diagnostic procedures. To this end, a three-in-one primary construct was designed from which three secondary constructs can be derived each capable of employing either type I, type II secretory or cytoplasmic pathways. Protein expression and secretion were performed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blotting. To further enhance protein secretion, the effect of several controllable chemical factors including IPTG, Triton X-100, sucrose, and glycine were individually investigated at the outset. In the next step, according to a fractional factorial approach, the synergistic effects of IPTG, Triton X-100, and glycine on secretion were further investigated. To ascertain the structure and function of the secreted recombinant proteins, dynamic light scattering was utilized to confirm the rPF4 tetramerization and heparin-mediated ultra-large complex formation. Moreover, Raman spectroscopy and Western blotting were exploited to evaluate the secondary and quaternary structures, respectively. The type II secretory pathway was proven to be superior to type I in the case of rPF4 secretion. Supplementation with chemical enhancers improved the protein secretion mediated by the Type II system to approximately more than 500 μg/mL. Large quantities of native rPF4 up to 20 mg were purified as the culture medium was scaled up to 40 mL. Western blotting confirmed the formation of dimers and tetramers in the secreted rPF4 proteins. Dynamic light scattering revealed the rPF4 oligomerization into of larger complexes of approximately 100–1200 nm in size following heparin supplementation, implying proper protein folding and tetramerization. Moreover, the rPF4 secondary structure was found to be 43.5% Random coil, 32.5% β-sheet, 18.6% α-helix and 4.9% Turn, which is in perfect agreement with the native structure. Our results indicate that the gram-negative type II bacterial secretory system holds a great promise as a reliable protein production strategy with industrial applications. However, further efforts are required to realize the full potential of secretory pathways regarding their application to proteins with distinct characteristics.

BamA is required for autotransporter secretion

BackgroundIn Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the “passenger” domain) and a β-barrel that aids its export. While it is known that the folding and insertion of the β-barrel domain utilize the β-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted β-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the β-barrel domain of the autotransporter. MethodsTo ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA. ResultsWe observed that each protein's β-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's β-barrel is more than that through the BamA β-barrel. ConclusionsSecretion of autotransporters most likely occurs through an incompletely formed β-barrel domain of the autotransporter in conjunction with BamA. General significanceSecretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.

Ultrastructure of intracytoplasmic Rickettsia-like infection of the gills of the teleost Archosargus probatocephalus (Sparidae) in northeastern Brazil.

A histopathological survey was conducted to investigate the presence of microparasites in fish Archosargus probatocephalus in a river near Maceió, Brazil. Light microscope observations of fragments of gill showed the presence of small cysts containing numerous myxospores that were morphologically identified as Henneguya. Transmission electron microscopy observations further revealed several gill cells containing groups of prokaryotic cells within large cytoplasmic vacuoles. Each infected host cell displayed a single vacuole containing a variable number of Rickettsia-like cells (up to 11), some of which presented the dumbbell shape characteristic of binary fission. The Rickettsia-like cells were pleomorphic, without a nucleus and with chromatin dispersed in the cytoplasm. They had a thin electron-dense wall of Gram-negative type. The morphology of these prokaryotic was similar to those of the order Rickettsiales and was described as a Rickettsia-like organism. Histopathological evaluation showed that several vacuole membranes had a lysed appearance. Some had ruptured, thus allowing direct contact between the Rickettsia-like organism and the cytoplasm of the host cell. The rupturing of the branchial epithelium may have contributed towards reduction of the surface area of the gills, but it is not possible to say that this was the cause of the host's death.

Aminoglycosides: Time for the Resurrection of a Neglected Class of Antibacterials?

This Viewpoint addresses the question of whether, after years of declining use, the time is ripe for renewed investigations into the aminoglycoside class of antibiotics for the development of novel therapeutic agents for the treatment of drug-resistant bacterial infections, particularly of the Gram-negative type. The reasons underlying the decline in use of the aminoglycosides are briefly considered and found to be outweighed by the ever-increasing clinical need for improved antibacterials with which to combat modern day multidrug resistant pathogens. The potential of the aminoglycosides builds on their well-established pharmacokinetics/pharmacodynamics (PK/PD), mechanisms of action, toxicity, and resistance and the extensive existing structure-activity relationship (SAR) databases, which permit rational, informed drug design. When coupled with the power of modern synthetic organic chemistry and improved funding scenarios, these multiple attributes open the door for the development of structurally novel, potent, and less toxic aminoglycosides to address the pressing societal problem of multidrug-resistant (MDR) infectious disease.

The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the Shigella type III secretion system

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial "minicells" have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of "pods" with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiGC), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiGC T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.

DeepT3: deep convolutional neural networks accurately identify Gram-negative bacterial type III secreted effectors using the N-terminal sequence.

Various bacterial pathogens can deliver their secreted substrates also called effectors through Type III secretion systems (T3SSs) into host cells and cause diseases. Since T3SS secreted effectors (T3SEs) play important roles in pathogen-host interactions, identifying them is crucial to our understanding of the pathogenic mechanisms of T3SSs. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to develop a novel and effective method to screen and select putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. We develop a deep convolution neural network to directly classify any protein sequence into T3SEs or non-T3SEs, which is useful for both effector prediction and the study of sequence-function relationship. Different from traditional machine learning-based methods, our method automatically extracts T3SE-related features from a protein N-terminal sequence of 100 residues and maps it to the T3SEs space. We train and test our method on the datasets curated from 16 species, yielding an average classification accuracy of 83.7% in the 5-fold cross-validation and an accuracy of 92.6% for the test set. Moreover, when comparing with known state-of-the-art prediction methods, the accuracy of our method is 6.31-20.73% higher than previous methods on a common independent dataset. Besides, we visualize the convolutional kernels and successfully identify the key features of T3SEs, which contain important signal information for secretion. Finally, some effectors reported in the literature are used to further demonstrate the application of DeepT3. DeepT3 is freely available at: https://github.com/lje00006/DeepT3. Supplementary data are available at Bioinformatics online.

ANTIBACTERIAL AND ANTIBIOFILM EFFECTS OF BEE VENOM FROM (APIS MELLIFERA) ON MULTIDRUG-RESISTANT BACTERIA (MDRB)

The prevalence of (MDRB) is increasing worldwide; therefore, this study aimed to identify the most common MDRB in clinical specimens and meet the urgentneed to develop new antibacterial drugs to control their intractable infection. Additionally, due to the confrontation of the infection associated with bacterial biofilms, which are difficult to treat, and cause problems to public health, which require real solutions. Bee Venom produced by the glands of (Apis mellifera) is a complex mixture of active peptides, enzymes, and amines. So, it is considered a fertile environment for research to achieve the goal of this study. The results of the specimen’s examination showed that, from a total of 500 clinical specimens, there are 224 specimens exhibited no growth, while 276 were positive. From 276 positive cultures, 317 isolates were obtained. Out of the 317 bacterial isolates, 169 (53.3%) were Gram-negative bacteria (GNB), and 148 (46.7%) were Gram-positive (GP). It was of this number 124 (39.1%) were multidrug-resistant (MDR) isolates of which 89 (71.77%) were Gram-negative type, including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa and 35 (28.23%) were Gram-positive including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Vancomycin-resistant Staphylococcus aureus, Staphylococcus haemolyticus and Enterococcus faecalis. Antibacterial assays showed that Bee Venom possesses strong potential effect against MDR isolates including both GNB and GPB.with a wide range of MICs and MLCs concentration-spacing between 3.125 – 50 μg/mL and 6.25 – 100 μg/mL, respectively against all MDR-GNB and GPB. It was found that GPB was more sensitive at lower concentrations of Bee Venom than GNB. In addition, Bee Venom sub-MICs values against the most biofilm bacterial produces namely; E. faecalis, P. aeruginosa, S. aureus, VRSA, S. haemolyticus and E. faecalis exhibited sharp reduction in their biofilms ranged between (63.8- 92%) especially at ½ MICs according to each bacterium, exclude E. faecalis biofilm was moderately affected (39%). While, at another tested sub-MICs showed moderate, weak, and no antibiofilm effects.

The Chlamydia trachomatis PmpD adhesin forms higher order structures through disulphide-mediated covalent interactions.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen, and the leading cause of infectious blindness worldwide. We have recently shown that immunization with the highly conserved antigenic passenger domain of recombinant Ct polymorphic membrane protein D (rPmpD) is protective in the mouse model of Ct genital tract infection, and previously, that ocular anti-rPmpD antibodies are elicited following vaccination. However, the mechanisms governing the assembly and structure-function relationship of PmpD are unknown. Here, we provide a biophysical analysis of this immunogenic 65 kDa passenger domain fragment of PmpD. Using differential cysteine labeling coupled with LC-MS/MS analysis, we show that widespread intra- and intermolecular disulphide interactions play important roles in the preservation of native monomeric secondary structure and the formation of higher-order oligomers. While it has been proposed that FxxN and GGA(I, L,V) repeat motifs in the Pmp21 ortholog in Chlamydia pneumoniae mediate self-interaction, no such role has previously been identified for cysteine residues in chlamydial Pmps. Further characterisation reveals that oligomeric proteoforms and rPmpD monomers adopt β–sheet folds, consistent with previously described Gram-negative bacterial type V secretion systems (T5SSs). We also highlight adhesin-like properties of rPmpD, showing that both soluble rPmpD and anti-rPmpD serum from immunized mice abrogate binding of rPmpD-coated beads to mammalian cells in a dose-dependent fashion. Hence, our study provides further evidence that chlamydial Pmps may function as adhesins, while elucidating yet another important mechanism of self-association of bacterial T5SS virulence factors that may be unique to the Chlamydiaceae.