Microanatomy of the antennal glands and their Na+/K+-ATPase activity in three true crab species (Brachyura), Portunus pelagicus (Linnaeus, 1758) (Portunidae), Macrophthalmus dentipes Lucas in Guérin, 1836 (Macrophthalmidae) and Eriocheir hepuensis Dai, 1991 (Varunidae)

There are differences between various crab species in the function and structure of organs involved in ionic and osmotic regulation processes. The antennal glands together constitute one of the most important organs involved in the osmoregulation in crabs. The present investigation aimed to study the tissue structure of the antennal glands and their Na+/K+-ATPase (NKA) pump activity in three true crab species from three different habitats, including the marine (Portunus pelagicus (Linnaeus, 1758)), the estuarine (Macrophthalmus dentipes Lucas in Guérin, 1836) and the freshwater habitat (Eriocheir hepuensis Dai, 1991). In this regard, the tissue structure of the antennal glands and the activity of the Na+/K+-ATPase (NKA) pump were assessed in these three selected species. The results showed that the antennal glands in all studied species consisted of two anterior parts and a posterior part. The anterior parts are composed of the proximal tubular region (PT) and the distal tubular region (DT). The PT and DT parts comprised the coelomosac and labyrinths in the anterior portion, and the bladder located in the posterior portion. However, despite the similarity in the general tissue structure of the antennal gland in marine, estuarine and freshwater crab species, some structural differences were observed between those species. Labyrinth cells, coelomosac podocytes, and bladder cells in the estuarine crab M. dentipes contained large vacuoles especially on the top (i.e., near the lumen) of the cells. The highest amount of NKA pump activity was measured in the antennal glands of M. dentipes (). The NKA pump plays a more important role in the estuary and seawater adaptation of crabs, but freshwater species are not highly dependent on the NKA enzyme for osmoregulation.

Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

Overexpression of the adeB Efflux Pump Gene in Tigecycline-Resistant Acinetobacter baumannii Clinical Isolates and Its Inhibition by (+)Usnic Acid as an Adjuvant

Acinetobacter species are among the most life-threatening Gram-negative bacilli, causing hospital-acquired infections, and they are associated with high morbidity and mortality. They show multidrug resistance that acts via various mechanisms. In Acinetobacter baumannii, efflux pump-mediated resistance to many antimicrobial compounds, including tigecycline, has been widely reported. Natural compounds have been used for their various pharmacological properties, including anti-efflux pump activity. The present study aimed to evaluate the efflux pump-mediated resistance mechanism of Acinetobacter baumannii and the effect of (+)Usnic acid as an efflux pump inhibitor with tigecycline. For detecting the efflux pump activity of tigecycline-resistant Acinetobacter baumannii isolates, microbroth dilution method and real-time quantitative reverse transcription–polymerase chain reaction was used. (+)Usnic acid was added to tigecycline and tested by the checkerboard method to evaluate its efficacy as an efflux pump inhibitor. qRT-PCR analysis was carried out to show the downregulation of the efflux pump in the isolates. Out of 42 tigecycline-resistant Acinetobacter baumannii isolates, 19 showed efflux pump activity. All 19 strains expressed the adeB gene. (+)Usnic acid as an adjuvant showed better efficacy in lowering the minimum inhibitory concentration compared with the conventional efflux pump inhibitor, carbonyl cyanide phenylhydrazone.

New Trimethoprim-Like Molecules: Bacteriological Evaluation and Insights into Their Action

This work reports a detailed characterization of the antimicrobial profile of two trimethoprim-like molecules (compounds 1a and 1b) identified in previous studies. Both molecules displayed remarkable antimicrobial activity, particularly when combined with sulfamethoxazole. In disk diffusion assays on Petri dishes, compounds 1a and 1b showed synergistic effects with colistin. Specifically, in combinations with low concentrations of colistin, very large increases in the activities of compounds 1a and 1b were determined, as demonstrated by alterations in the kinetics of bacterial growth despite only slight changes in the fractional inhibitory concentration index. The effect of colistin may be to increase the rate of antibiotic entry while reducing efflux pump activity. Compounds 1a and 1b were susceptible to extrusion by efflux pumps, whereas the inhibitor phenylalanine arginyl β-naphthylamide (PAβN) exerted effects similar to those of colistin. The interactions between the target enzyme (dihydrofolate reductase), the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), and the studied molecules were explored using enzymology tools and computational chemistry. A model based on docking results is reported.

Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

Since dysregulation of intracellular calcium (Ca2+) levels is a common occurrence in neurodegenerative diseases, including Alzheimer’s disease (AD), the study of proteins that can correct neuronal Ca2+ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca2+-ATPase (PMCA), a high-affinity Ca2+ pump, is functionally impaired in AD and is inhibited by amyloid-β peptide (Aβ) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca2+-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and other modulators of intracellular Ca2+ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aβ and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aβ and tau on the pump, and counteracts the neurotoxicity of Aβ and tau by interacting with them.

The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

EVALUATION OF EFFLUX PUMP ACTIVITY AND BIOFILM FORMATION IN MULTIDRUG RESISTANT KLEBSIELLA PNEUMONIAE ISOLATES IN TANTA, EGYPT

Nosocomial and community acquired infections that caused by multidrug-resistant (MDR) Klebsiella pneumoniae isolates are widespread recently resulting in high morbidity and mortality due to limited number of treatment options with effective antibiotics. The aim of this study is to evaluate the antibiotic resistance profile, biofilm formation and efflux pump activity of MDR K. pneumoniae isolates collected from different hospitals in Tanta, Egypt. A total of 70 K. pneumoniae isolates characterized by standard biochemical tests and confirmed by MALDI-TOF/MS were screened for antibiotic susceptibility, efflux pump activity and biofilm formation. Isolates displayed high resistance to penicillins, cephalosporins, trimethoprim-sulfamethoxazole and the majority of tested fluoro/-quinolones and decreased resistance to imipenem, amikacin, chloramphenicol, tigecycline and colistin. Out of 70 K. pneumoniae isolates, 2 isolates exhibited Pan Drug-Resistance (PDR) profile while 57 (81.4%) and 11 (15.7%) exhibited MDR and Extensively drug-resistance (XDR) profiles, respectively. Sixty-four (91.4%) isolates exhibited efflux pump activity while all tested isolates had the ability to form biofilm with varied degrees as 40 (57.1%), 26 (37.1%), and 4 (5.7%) isolates were strong, moderate and weak biofilm producers, respectively. Also, a strong relation between efflux pump activity and biofilm formation per isolate was detected. In conclusion, Multidrug resistance, biofilm formation and efflux pump capabilities in K. pneumoniae have serious public health implications in the management and control of infections caused by this bacterium. Therefore, a multifaceted approach and precise planning are recommended in controlling these infections

Molecular and phenotypic characterization of efflux pump and biofilm in multi-drug resistant non-typhoidal Salmonella Serovars isolated from food animals and handlers in Lagos Nigeria

Background Multidrug resistance efflux pumps and biofilm formation are mechanisms by which bacteria can evade the actions of many antimicrobials. Antibiotic resistant non-typhoidal Salmonella serovars have become wide spread causing infections that result in high morbidity and mortality globally. The aim of this study was to evaluate the efflux pump activity and biofilm forming capability of multidrug resistant non-typhoidal Salmonella (NTS) serovars isolated from food handlers and animals (cattle, chicken and sheep) in Lagos. Methods Forty eight NTS serovars were subjected to antibiotic susceptibility testing by the disc diffusion method and phenotypic characterization of biofilm formation was done by tissue culture plate method. Phenotypic evaluation of efflux pump activity was done by the ethidium bromide cartwheel method and genes encoding biofilm formation and efflux pump activity were determined by PCR. Results All 48 Salmonella isolates displayed resistance to one or more classes of test antibiotics with 100% resistance to amoxicillin-clavulanic acid. Phenotypically, 28 (58.3%) of the isolates exhibited efflux pump activity. However, genotypically, 7 (14.6%) of the isolates harboured acrA, acrB and tolC, 8 (16.7%) harboured acrA, acrD and tolC while 33 (68.8%) possessed acrA, acrB, acrD and tolC. All (100%) the isolates phenotypically had the ability to form biofilm with 23 (47.9%), 24 (50.0%), 1 (2.1%) categorized as strong (SBF), moderate (MBF) and weak (WBF) biofilm formers respectively but csgA gene was detected in only 23 (47.9%) of them. Antibiotic resistance frequency was significant (p < 0.05) in SBF and MBF and efflux pump activity was detected in 6, 21, and 1 SBF, MBF and WBF respectively. Conclusion These data suggest that Salmonella serovars isolated from different food animals and humans possess active efflux pumps and biofilm forming potential which has an interplay in antibiotic resistance. There is need for prudent use of antibiotics in veterinary medicine and scrupulous hygiene practice to prevent the transmission of multidrug resistant Salmonella species within the food chain.