Merkel Cell Polyomavirus in the Context of Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders.

Despite recent advances in prevention, detection and treatment, oral squamous cell carcinoma (OSCC) remains a global health concern, strongly associated with environmental and lifestyle risk factors and infection with oncogenic viruses. Merkel Cell Polyomavirus (MCPyV), well known to be the causative agent of Merkel Cell Carcinoma (MCC) has been found in OSCC, suggesting its potential role as a co-factor in the development of oral cavity cancers. To improve our understanding about MCPyV in oral cavities, the detection and analysis of MCPyV DNA, transcripts and miRNA were performed on OSCCs and oral potentially malignant disorders (OPMDs). In addition, the cellular miR-375, known to be deregulated in tumors, was examined. MCPyV DNA was found in 3 out of 11 OSCC and 4 out of 12 OPMD samples, with a viral mean value of 1.49 × 102 copies/mL. Viral integration was not observed and LTAg and VP1 transcripts were detected. Viral miRNAs were not detected whereas the cellular miR-375 was found over expressed in all MCPyV positive oral specimens. Our results reported evidence of MCPyV replication in both OSCC and OPMD suggesting the oral cavity as a site of replicative MCPyV infection, therefore underscoring an active role of this virus in the occurrence of oral lesions.

The Shigella flexneri virulence factor apyrase is released inside eukaryotic cells to hijack host cell fate.

IMPORTANCE: In this paper, we demonstrated that apyrase is released within the host cell cytoplasm during infection to target the intracellular ATP pool. By degrading intracellular ATP, apyrase contributes to prevent caspases activation, thereby inhibiting the activation of pyroptosis in infected cells. Our results show, for the first time, that apyrase is involved in the modulation of host cell survival, thereby aiding this pathogen to dampen the inflammatory response. This work adds a further piece to the puzzle of Shigella pathogenesis. Due to its increased spread worldwide, prevention and controlling strategies are urgently needed. Overall, this study highlighted apyrase as a suitable target for an anti-virulence therapy to tackle this pathogen.

Insightful Improvement in the Design of Potent Uropathogenic E. coli FimH Antagonists.

Selective antiadhesion antagonists of Uropathogenic Escherichia coli (UPEC) type-1 Fimbrial adhesin (FimH) are attractive alternatives for antibiotic therapies and prophylaxes against acute or recurrent urinary tract infections (UTIs) caused by UPECs. A rational small library of FimH antagonists based on previously described C-linked allyl α-D-mannopyranoside was synthesized using Heck cross-coupling reaction using a series of iodoaryl derivatives. This work reports two new members of FimH antagonist amongst the above family with sub nanomolar affinity. The resulting hydrophobic aglycones, including constrained alkene and aryl groups, were designed to provide additional favorable binding interactions with the so-called FimH "tyrosine gate". The newly synthesized C-linked glycomimetic antagonists, having a hydrolytically stable anomeric linkage, exhibited improved binding when compared to previously published analogs, as demonstrated by affinity measurement through interactions by FimH lectin. The crystal structure of FimH co-crystallized with one of the nanomolar antagonists revealed the binding mode of this inhibitor into the active site of the tyrosine gate. In addition, selected mannopyranoside constructs neither affected bacterial growth or cell viability nor interfered with antibiotic activity. C-linked mannoside antagonists were effective in decreasing bacterial adhesion to human bladder epithelial cells (HTB-9). Therefore, these molecules constituted additional therapeutic candidates' worth further development in the search for potent anti-adhesive drugs against infections caused by UPEC.

Inflammatory Response in Caco-2 Cells Stimulated with Anisakis Messengers of Pathogenicity.

Background: Anisakis spp. third-stage larvae (L3) are the causative agents of human zoonosis called anisakiasis. The accidental ingestion of L3 can cause acute and chronic inflammation at the gastric, intestinal, or ectopic levels. Despite its relevance in public health, studies on pathogenetic mechanisms and parasite-human interplay are scarce. The aim of this study was to investigate the human inflammatory response to different Anisakis vehicles of pathogenicity. Methods: Human colorectal adenocarcinoma (Caco-2) cells were exposed to Anisakis L3 (the initial contact with the host), extracellular vesicles (EVs, Anisakis-host communication), and crude extract (CE, the larval dying). The protein quantity and gene expression of two pro-inflammatory cytokines (IL-6 and IL-8) were investigated using an ELISA test (6 h and 24 h) and a qReal-Time PCR (1 h, 6 h, and 24 h), respectively. Results: The L3 and EVs induced a downregulation in both the Il-6 and Il-8 gene expression and protein quantity. On the contrary, the CE stimulated IL-6 gene expression and its protein release, not affecting IL-8. Conclusions: The Caco-2 cells seemed to not react to the exposure to the L3 and EVs, suggesting a parasite's immunomodulating action to remain alive in an inhospitable niche. Conversely, the dying larva (CE) could induce strong activation of the immune strategy of the host that, in vivo, would lead to parasite expulsion, eosinophilia, and/or granuloma formation.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion / Estrategias adaptativas de Escherichia coli CFT073 uropatógena: desde el crecimiento en medios de laboratorio hasta la virulencia durante la adhesión de la célula huésped

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.(AU)

Genetic Diversity of Antimicrobial Resistance and Key Virulence Features in Two Extensively Drug-Resistant Acinetobacter baumannii Isolates.

In recent decades, Acinetobacter baumannii emerged as a major infective menace in healthcare settings due to scarce therapeutic options to treat infections. Therefore, undertaking genome comparison analyses of multi-resistant A. baumannii strains could aid the identification of key bacterial determinants to develop innovative anti-virulence approaches. Following genome sequencing, we performed a molecular characterization of key genes and genomic comparison of two A. baumannii strains, #36 and #150, with selected reference genomes. Despite a different antibiotic resistance gene content, the analyzed strains showed a very similar antibiogram profile. Interestingly, the lack of some important virulence determinants (i.e., bap, ata and omp33-36) did not abrogate their adhesive abilities to abiotic and biotic surfaces, as reported before; indeed, strains retained these capacities, although to a different extent, suggesting the presence of distinct vicarious genes. Conversely, secretion systems, lipopolysaccharide (LPS), capsule and iron acquisition systems were highly similar to A. baumannii reference strains. Overall, our analyses increased our knowledge on A. baumannii genomic content and organization as well as the genomic events occurring in nosocomial isolates to better fit into changing healthcare environments.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion.

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.

The Acinetobacter baumannii model can explain the role of small non-coding RNAs as potential mediators of host-pathogen interactions.

Bacterial small RNAs (sRNAs) research has accelerated over the past decade, boosted by advances in RNA-seq technologies and methodologies for capturing both protein-RNA and RNA-RNA interactions. The emerging picture is that these regulatory sRNAs play important roles in controlling complex physiological processes and are required to survive the antimicrobial challenge. In recent years, the RNA content of OMVs/EVs has also gained increasing attention, particularly in the context of infection. Secreted RNAs from several bacterial pathogens have been characterized but the exact mechanisms promoting pathogenicity remain elusive. In this review, we briefly discuss how secreted sRNAs interact with targets in infected cells, thus representing a novel perspective of host cell manipulation during bacterial infection. During the last decade, Acinetobacter baumannii became clinically relevant emerging pathogens responsible for nosocomial and community-acquired infections. Therefore, we also summarize recent findings of regulation by sRNAs in A. baumannii and discuss how this emerging bacterium utilizes many of these sRNAs to adapt to its niche and become successful human pathogen.

Gram-Negative Bacteria Holding Together in a Biofilm: The Acinetobacter baumannii Way.

Bacterial biofilms are a serious public-health problem worldwide. In recent years, the rates of antibiotic-resistant Gram-negative bacteria associated with biofilm-forming activity have increased worrisomely, particularly among healthcare-associated pathogens. Acinetobacter baumannii is a critically opportunistic pathogen, due to the high rates of antibiotic resistant strains causing healthcare-acquired infections (HAIs). The clinical isolates of A. baumannii can form biofilms on both biotic and abiotic surfaces; hospital settings and medical devices are the ideal environments for A. baumannii biofilms, thereby representing the main source of patient infections. However, the paucity of therapeutic options poses major concerns for human health infections caused by A. baumannii strains. The increasing number of multidrug-resistant A. baumannii biofilm-forming isolates in association with the limited number of biofilm-eradicating treatments intensify the need for effective antibiofilm approaches. This review discusses the mechanisms used by this opportunistic pathogen to form biofilms, describes their clinical impact, and summarizes the current and emerging treatment options available, both to prevent their formation and to disrupt preformed A. baumannii biofilms.

Acinetobacter baumannii: An Ancient Commensal with Weapons of a Pathogen.

Acinetobacter baumannii is regarded as a life-threatening pathogen associated with community-acquired and nosocomial infections, mainly pneumonia. The rise in the number of A. baumannii antibiotic-resistant strains reduces effective therapies and increases mortality. Bacterial comparative genomic studies have unraveled the innate and acquired virulence factors of A. baumannii. These virulence factors are involved in antibiotic resistance, environmental persistence, host-pathogen interactions, and immune evasion. Studies on host-pathogen interactions revealed that A. baumannii evolved different mechanisms to adhere to in order to invade host respiratory cells as well as evade the host immune system. In this review, we discuss current data on A. baumannii genetic features and virulence factors. An emphasis is given to the players in host-pathogen interaction in the respiratory tract. In addition, we report recent investigations into host defense systems using in vitro and in vivo models, providing new insights into the innate immune response to A. baumannii infections. Increasing our knowledge of A. baumannii pathogenesis may help the development of novel therapeutic strategies based on anti-adhesive, anti-virulence, and anti-cell to cell signaling pathways drugs.

Urinary tract infections: Can we prevent uropathogenic Escherichia coli infection with dietary intervention?

Urinary tract infections (UTIs) are among the most common causes of infections in women. Via the fecal-perineal-urethral route, uropathogenic Escherichia coli (UPEC) can cause ascending urinary tract infections, including cystitis and pyelonephritis. These infections re-occur within six months or they account for, at least, three episodes within a year of recurrent UTIs (rUTIs). Long term and continuous antibiotic treatment or prophylaxis should be considered as the last options in rUTIs. Conversely, updated European Association of Urology guidelines recommend non-antimicrobial approaches to prevent rUTIs. Accordingly, several studies reported the efficacy of number of natural molecules in inhibiting UPEC adhesion to bladder cells, restraining bacterial growth, as well as stimulating the host innate immune defenses, and protecting the bladder and the kidney mucosa. Therefore, we propose an "anti-UPEC" diet enriched of foods containing natural compounds that were proven effective against UPEC, such as D-mannose, cranberry extracts and medicinal plants. Being a valuable and safe clinical approach to reduce UTI recurrence and limiting the detrimental effects of long and continuous antibiotic prophylaxis, dietary interventions should be evaluated in future clinical trials.

SARS-CoV-2: Comparative analysis of different RNA extraction methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

Acinetobacter baumannii Targets Human Carcinoembryonic Antigen-Related Cell Adhesion Molecules (CEACAMs) for Invasion of Pneumocytes.

Multidrug-resistant Acinetobacter baumannii is regarded as a life-threatening pathogen mainly associated with nosocomial and community-acquired pneumonia. Here, we show that A. baumannii can bind the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors CEACAM1, CEACAM5, and CEACAM6. This specific interaction enhances A. baumannii internalization in membrane-bound vacuoles, promptly decorated with Rab5, Rab7, and lipidated microtubule-associated protein light chain 3 (LC3). Dissecting intracellular signaling pathways revealed that infected pneumocytes trigger interleukin-8 (IL-8) secretion via the extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) signaling pathways for A. baumannii clearance. However, in CEACAM1-L-expressing cells, IL-8 secretion lasts only 24 h, possibly due to an A. baumannii-dependent effect on the CEACAM1-L intracellular domain. Conversely, the glycosylphosphatidylinositol-anchored CEACAM5 and CEACAM6 activate the c-Jun NH2-terminal kinase (JNK)1/2-Rubicon-NOX2 pathway, suggestive of LC3-associated phagocytosis. Overall, our data show for the first time novel mechanisms of adhesion to and invasion of pneumocytes by A. baumannii via CEACAM-dependent signaling pathways that eventually lead to bacterial killing. These findings suggest that CEACAM upregulation could put patients at increased risk of lower respiratory tract infection by A. baumannii IMPORTANCE This work shows for the first time that Acinetobacter baumannii binds to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5, and CEACAM6. This binding significantly enhances A. baumannii internalization within alveolar host cell epithelia. Intracellular trafficking involves typical Rab5 and Rab7 vacuolar proteins as well as light chain 3 (LC3) and slowly progresses to bacterial killing by endosome acidification. CEACAM engagement by A. baumannii leads to distinct and specific downstream signaling pathways. The CEACAM1 pathway finely tunes interleukin-8 (IL-8) secretion, whereas CEACAM5 and CEACAM6 mediate LC3-associated phagocytosis. The present study provides new insights into A. baumannii-host interactions and could represent a promising therapeutic strategy to reduce pulmonary infections caused by this pathogen.

Fecal microRNAs as Innovative Biomarkers of Intestinal Diseases and Effective Players in Host-Microbiome Interactions.

Over the past decade, short non-coding microRNAs (miRNAs), including circulating and fecal miRNAs have emerged as important modulators of various cellular processes by regulating the expression of target genes. Recent studies revealed the role of miRNAs as powerful biomarkers in disease diagnosis and for the development of innovative therapeutic applications in several human conditions, including intestinal diseases. In this review, we explored the literature and summarized the role of identified dysregulated fecal miRNAs in intestinal diseases, with particular focus on colorectal cancer (CRC) and celiac disease (CD). The aim of this review is to highlight one fascinating aspect of fecal miRNA function related to gut microbiota shaping and bacterial metabolism influencing. The role of miRNAs as "messenger" molecules for inter kingdom communications will be analyzed to highlight their role in the complex host-bacteria interactions. Moreover, whether fecal miRNAs could open up new perspectives to develop novel suitable biomarkers for disease detection and innovative therapeutic approaches to restore microbiota balance will be discussed.

The Global Emergency of Novel Coronavirus (SARS-CoV-2): An Update of the Current Status and Forecasting.

Over the past two decades, there have been two major outbreaks where the crossover of animal Betacoronaviruses to humans has resulted in severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). In December 2019, a global public health concern started with the emergence of a new strain of coronavirus (SARS-CoV-2 or 2019 novel coronavirus, 2019-nCoV) which has rapidly spread all over the world from its origin in Wuhan, China. SARS-CoV-2 belongs to the Betacoronavirus genus, which includes human SARS-CoV, MERS and two other human coronaviruses (HCoVs), HCoV-OC43 and HCoV-HKU1. The fatality rate of SARS-CoV-2 is lower than the two previous coronavirus epidemics, but it is faster spreading and the large number of infected people with severe viral pneumonia and respiratory illness, showed SARS-CoV-2 to be highly contagious. Based on the current published evidence, herein we summarize the origin, genetics, epidemiology, clinical manifestations, preventions, diagnosis and up to date treatments of SARS-CoV-2 infections in comparison with those caused by SARS-CoV and MERS-CoV. Moreover, the possible impact of weather conditions on the transmission of SARS-CoV-2 is also discussed. Therefore, the aim of the present review is to reconsider the two previous pandemics and provide a reference for future studies as well as therapeutic approaches.

FimH and Anti-Adhesive Therapeutics: A Disarming Strategy Against Uropathogens.

Chaperone-usher fimbrial adhesins are powerful weapons against the uropathogens that allow the establishment of urinary tract infections (UTIs). As the antibiotic therapeutic strategy has become less effective in the treatment of uropathogen-related UTIs, the anti-adhesive molecules active against fimbrial adhesins, key determinants of urovirulence, are attractive alternatives. The best-characterized bacterial adhesin is FimH, produced by uropathogenic Escherichia coli (UPEC). Hence, a number of high-affinity mono- and polyvalent mannose-based FimH antagonists, characterized by different bioavailabilities, have been reported. Given that antagonist affinities are firmly associated with the functional heterogeneities of different FimH variants, several FimH inhibitors have been developed using ligand-drug discovery strategies to generate high-affinity molecules for successful anti-adhesion therapy. As clinical trials have shown d-mannose's efficacy in UTIs prevention, it is supposed that mannosides could be a first-in-class strategy not only for UTIs, but also to combat other Gram-negative bacterial infections. Therefore, the current review discusses valuable and effective FimH anti-adhesive molecules active against UTIs, from design and synthesis to in vitro and in vivo evaluations.

d-Mannose Treatment neither Affects Uropathogenic Escherichia coli Properties nor Induces Stable FimH Modifications.

Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC). Acute and recurrent UTIs are commonly treated with antibiotics, the efficacy of which is limited by the emergence of antibiotic resistant strains. The natural sugar d-mannose is considered as an alternative to antibiotics due to its ability to mask the bacterial adhesin FimH, thereby preventing its binding to urothelial cells. Despite its extensive use, the possibility that d-mannose exerts "antibiotic-like" activity by altering bacterial growth and metabolism or selecting FimH variants has not been investigated yet. To this aim, main bacterial features of the prototype UPEC strain CFT073 treated with d-mannose were analyzed by standard microbiological methods. FimH functionality was analyzed by yeast agglutination and human bladder cell adhesion assays. Our results indicate that high d-mannose concentrations have no effect on bacterial growth and do not interfere with the activity of different antibiotics. d-mannose ranked as the least preferred carbon source to support bacterial metabolism and growth, in comparison with d-glucose, d-fructose, and l-arabinose. Since small glucose amounts are physiologically detectable in urine, we can conclude that the presence of d-mannose is irrelevant for bacterial metabolism. Moreover, d-mannose removal after long-term exposure did not alter FimH's capacity to bind to mannosylated proteins. Overall, our data indicate that d-mannose is a good alternative in the prevention and treatment of UPEC-related UTIs.

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria.

BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.

Insights into the Periplasmic Proteins of Acinetobacter baumannii AB5075 and the Impact of Imipenem Exposure: A Proteomic Approach.

Carbapenem-resistant Acinetobacter baumannii strains cause life-threatening infections due to the lack of therapeutic options. Although the main mechanisms underlying antibiotic-resistance have been extensively studied, the general response to maintain bacterial viability under antibiotic exposure deserves to be fully investigated. Since the periplasmic space contains several proteins with crucial cellular functions, besides carbapenemases, we decided to study the periplasmic proteome of the multidrug-resistant (MDR) A. baumannii AB5075 strain, grown in the absence and presence of imipenem (IMP). Through the proteomic approach, 65 unique periplasmic proteins common in both growth conditions were identified: eight proteins involved in protein fate, response to oxidative stress, energy metabolism, antibiotic-resistance, were differentially expressed. Among them, ABUW_1746 and ABUW_2363 gene products presented the tetratricopeptide repeat motif, mediating protein-protein interactions. The expression switch of these proteins might determine specific protein interactions to better adapt to changing environmental conditions. ABUW_2868, encoding a heat shock protein likely involved in protection against oxidative stress, was upregulated in IMP-exposed bacteria. Accordingly, the addition of periplasmic proteins from A. baumannii cultured with IMP increased bacterial viability in an antioxidant activity assay. Overall, this study provides the first insights about the composition of the periplasmic proteins of a MDR A. baumannii strain, its biological response to IMP and suggests possible new targets to develop alternative antibiotic drugs.