The Enhanced Expression of Cruzipain-Like Molecules in the Phytoflagellate Phytomonas serpens Recovered From the Invertebrate and Plant Hosts

Phytomonas serpens is a protozoan parasite that alternates its life cycle between two hosts: an invertebrate vector and the tomato fruit. This phytoflagellate is able to synthesize proteins displaying similarity to the cysteine peptidase named cruzipain, an important virulence factor from Trypanosoma cruzi, the etiologic agent of Chagas disease. Herein, the growth of P. serpens in complex medium (BHI) supplemented with natural tomato extract (NTE) resulted in the increased expression of cysteine peptidases, as verified by the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC and by gelatin-SDS-PAGE. Phytoflagellates showed no changes in morphology, morphometry and viability, but the proliferation was slightly reduced when cultivated in the presence of NTE. The enhanced proteolytic activity was accompanied by a significant increase in the expression of cruzipain-like molecules, as verified by flow cytometry using anti-cruzipain antibodies. In parallel, parasites incubated under chemically defined conditions (PBS supplemented with glucose) and added of different concentration of NTE revealed an augmentation in the production of cruzipain-like molecules in a typically dose-dependent way. Similarly, P. serpens recovered from the infection of mature tomatoes showed an increase in the expression of molecules homologous to cruzipain; however, cells showed a smaller size compared to parasites grown in BHI medium. Furthermore, phytoflagellates incubated with dissected salivary glands from Oncopeltus fasciatus or recovered from the hemolymph of infected insects also showed a strong enhance in the expression of cruzipain-like molecules that is more relevant in the hemolymph. Collectively, our results showed that cysteine peptidases displaying similarities to cruzipain are more expressed during the life cycle of the phytoflagellate P. serpens both in the invertebrate and plant hosts.

Dictamnine Inhibits the Adhesion to and Invasion of Uropathogenic Escherichia Coli (UPEC) to Urothelial Cells

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacteria associated with urinary tract infection (UTI). UPEC can cause UTI by adhering to and invading uroepithelial cells. Fimbriae is the most important virulence factor of UPEC, and a potentially promising target in developing novel antibacterial treatments. In this study, the antibacterial properties and effects of the compound dictamnine, extracted from the traditional Chinese medicine Cortex Dictamni, on the bacterial morphology, cell adhesion, and invasion of UPEC were studied. Dictamnine exhibited no obvious antibacterial activity against UPEC, but significantly impeded the ability of UPEC to adhere to and invade uroepithelial cells. RT-qPCR analysis showed that treatment downregulated the expression of type 1 fimbriae, P fimbriae, and curli fimbriae adhesion genes, and also downregulated adhesion-related receptor genes of uroepithelial cells. Transmission electron microscopy showed that dictamnine destroyed the structure of the fimbriae and the surface of the bacteria became smooth. These results suggest that dictamnine may help to prevent UTI by simultaneously targeting UPEC fimbriae and urothelial adhesin receptors, and may have a potential use as a new anti-UPEC drug.

H-NOX proteins in the virulence of pathogenic bacteria

Nitric oxide (NO) is a toxic gas encountered by bacteria as a product of their own metabolism or as a result of a host immune response. Non-toxic concentrations of NO have been shown to initiate changes in bacterial behaviors such as the transition between planktonic and biofilm-associated lifestyles. The heme nitric oxide/oxygen binding proteins (H-NOX) are a widespread family of bacterial heme-based NO sensors that regulate biofilm formation in response to NO. The presence of H-NOX in several human pathogens combined with the importance of planktonic-biofilm transitions to virulence suggests that H-NOX sensing may be an important virulence factor in these organisms. Here we review the recent data on H-NOX NO signaling pathways with an emphasis on H-NOX homologues from pathogens and commensal organisms. The current state of the field is somewhat ambiguous regarding the role of H-NOX in pathogenesis. However, it is clear that H-NOX regulates biofilm in response to environmental factors and may promote persistence in the environments that serve as reservoirs for these pathogens. Finally, the evidence that large subgroups of H-NOX proteins may sense environmental signals besides NO is discussed within the context of a phylogenetic analysis of this large and diverse family.

The VirB System Plays a Crucial Role in Brucella Intracellular Infection

Brucellosis is a highly prevalent zoonotic disease caused by Brucella. Brucella spp. are gram-negative facultative intracellular parasitic bacteria. Its intracellular survival and replication depend on a functional virB system, an operon encoded by VirB1–VirB12. Type IV secretion system (T4SS) encoded by the virB operon is an important virulence factor of Brucella. It can subvert cellular pathway and induce host immune response by secreting effectors, which promotes Brucella replication in host cells and induce persistent infection. Therefore, this paper summarizes the function and significance of the VirB system, focusing on the structure of the VirB system where VirB T4SS mediates biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV), the effectors of T4SS and the cellular pathways it subverts, which will help better understand the pathogenic mechanism of Brucella and provide new ideas for clinical vaccine research and development.

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.

Expression of the Type III Secretion System Genes in Epiphytic Erwinia amylovora Cells on Apple Stigmas Benefits Endophytic Infection at the Hypanthium

Erwinia amylovora causes fire blight on rosaceous plants. One of the major entry points of E. amylovora into hosts is flowers, where E. amylovora proliferates epiphytically on stigmatic and hypanthium surfaces and, subsequently, causes endophytic infection at the hypanthium. The type III secretion system (T3SS) is an important virulence factor in E. amylovora. Although the role of T3SS during endophytic infection is well characterized, its expression during epiphytic colonization and role in the subsequent infection is less understood. Here, we investigated T3SS gene expression in epiphytic E. amylovora on stigma and hypanthium of apple flowers under different relative humidities (RH). On stigma surfaces, T3SS was expressed in a high percentage of E. amylovora cells, and its expression promoted epiphytic growth. On hypanthium surfaces, however, T3SS was expressed in fewer E. amylovora cells than on the stigma, and displayed no correlation with epiphytic growth, even though T3SS expression is essential for infection. E. amylovora cells grown on stigmatic surfaces and then flushed down to the hypanthium displayed a higher level of T3SS expression than cells grown on the hypanthium surface alone. Furthermore, E. amylovora cells precultured on stigma had a higher potential to infect flowers than E. amylovora cells precultured in a T3SS-repressive medium. This suggests that T3SS induction during the stigmatic epiphytic colonization may be beneficial for subsequent infection. Finally, epiphytic expression of T3SS was influenced by RH. Higher percentage of stigmatic E. amylovora cells expressed T3SS under high RH than under low RH. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The Potency of NTHi lic1A Gene as a Biomarker in Determining The Severity of Post-Viral Acute Rhinosinusitis

BACKGROUND: The lic1A gene is an important virulence factor for non-typeable H. influenzae (NTHi), which allows its translocation from the nasopharynx into the sinonasal cavity and modulates more severe inflammatory processes. This study is aimed for identifying the potential correlation between the NTHi lic1A gene expressions and the severity of post-viral acute rhinosinusitis.METHODS: Sixty patients who were diagnosed with post-viral acute rhinosinusitis, were recruited from an ENT clinic in a referral hospital, in Bandung, West Java, Indonesia. All patients underwent a historical assessment and ENT examination. The nasal specimen was taken from the patient’s middle meatal. The NTHi lic1A gene expression was detected using Polymerase Chain Reaction (PCR).RESULTS: We observed that eight patients had the NTHi lic1A (+), with a strong correlation toward the dominant symptoms (nasal obstruction and discharge). In addition, the symptom’s duration of the NTHi lic1A (+) was twice longer than patients with the NTHi lic1A (-). Its severity was significantly more different between the two groups (p=0.034).CONCLUSION: Taken together, the presence of the NTHi lic1A gene is significantly associated with the severity of the disease and the symptom’s duration. Thus, the NTHi lic1A gene could potentially be a good marker for assessing the severity of post-viral acute rhinosinusitis cases in the future.KEYWORDS: H. influenzae, rhinosinusitis, nasal obstruction, virulence factors

Biofilm Producing Enterococcus Isolates from Vaginal Microbiota

Background: Enterococcus is an important cause of infection in the hospital as well as in the community. Methods: A prospective study was done in Medical College, Kolkata for a period of 2 years (from January 2018 to December 2019). After obtaining clearance from the Institutional Ethics Committee, Enterococcus isolates from cases of vaginitis were included in the study. Identification of Enterococcus species was done by Gram stain and conventional biochemical tests along with automated identification by VITEK 2 Compact. These isolates were tested for antimicrobial susceptibility to different antibiotics by Kirby Bauer disc diffusion method and minimum inhibitory concentration (MIC) by VITEK 2 Compact. Interpretation of susceptibility was done according to the Clinical and Laboratory Standards Institute (CLSI) 2017 guidelines. Biofilm detection for Enterococcus species was done. Results: During the period of 2 years, 39 isolates of Enterococcus spp. were obtained from vaginitis cases. Among these, 27 were Enterococcus faecalis and 12 Enterococcus faecium. All isolates were highly susceptible to vancomycin, teicoplanin, and linezolid. Biofilm was detected in eight isolates of which five were strong biofilm producer and three moderate biofilm producers. Conclusion: Biofilm production is an important virulence factor in Enterococcus isolates from vaginitis.

Molecular Detection of Some Capsulargenes of Klebsiella pneumoniaeIsolated from Clinical Samples

Klebsiella pneumoniae is an opportunistic pathogen that has been implicated as one of commonest cause of hospital and community acquired infections.The capsule of Klebsiella pneumoniae is an important virulence factor, involved in pathogenic mechanisms.So, this study aimed to isolateKlebsiella pneumoniae from different clinical samples from patients in Al-Diwaniyah teaching hospital and determine some virulence factors (capsulegenes)that used for serotyping of isolates. The study extended fromAugust to November 2020.A total of 31 isolates from 80 different clinical samples identified as Klebsiella pneumoniae by traditional biochemical tests ,Vitek system and 16SrRNA gene.The existenceof three genes from7tested capsulargenes wasdetectedby Polymerase chain reaction.The commonness serotype were k2,k54,k57 where K2 detected in 4 (12.9 %), K54 in 12 (38.7 %),k57 in 4 (12.9).But, the other capsular polysaccaride genes k1,k3,k5,k20not detectedin all isolates of this study.

The basis of α-hemolysis Negative Methicillin-resistant Staphylococcus Aureus Isolates from Beijing Children’s Hospital

Background. Methicillin-resistant Staphylococcus aureus (MRSA) Clonal Complex 59 (CC59) clone has spread among Chinese children, resulting in many Staphylococcus aureus infections. α-hemolysin (Hlα) is an important virulence factor of Staphylococcus aureus, but little research has been done on CC59 isolates with negative α-hemolysis. Results. During the 4 periods (2009-2011, 2012-2013, 2016, 2017), 291 MRSA isolates were collected. Isolates with β and δ hemolysis accounted for 60.47% among the MRSA isolates in 2009-2011; 56.41% in 2012-2013; 77.14% in 2016; and 56.25% in 2017. most ST59 isolates (94.38%), 9 ST338 isolates (100%) showed β and δ hemolysis, both ST59 and ST338 clone belong to CC59 clone. Twenty-two ST239 isolates (73.33%), 8 ST88 isolates (80%), 4 ST5 isolates (100%), 13 ST22 isolates (92.86%) and 6 ST398 isolates (85.71%) showed α and δ hemolysis. α hemolysin in most clinical isolates is highly conservative, each showed one amino acid locus variation, the most common mutation was threonine at position 275 instead of isoleucine, then glutamic acid replaced aspartic acid at 208. Seventeen ST59 and 2 ST338 isolates had no mutation, 3 ST59 isolates showed single mutation (C448G), and only one ST59 isolate showed multilocus mutation. Other ST typing, such as ST1, ST5, ST88, ST20, ST239 and ST398, all had multilocus mutations, sites were from 3 to 8, no conservative sequence was found among isolates with the same ST typing. The carrying rates of RNA III, Rot, agrA, SarR, SarU and SigB were all over 93%, the carrying rates of SarZ and SarA genes were 41.86% and 34.88% respectively. Trancriptional levels of hlα in isolates showed α and δ hemolysis and β and δ hemolysis were equal. USA300 and R23 produced Hlα, R23 didn’t showed α hemolysis phenotype.Conclusions. Most clinical CC59 isolates from children in China were α hemolysis negative. There was no statistically significant difference in hlα gene and RNA expression, they produced the protein. The reason for the phenotypic deletion probably related to β hemolysin (Hlβ).