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Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD). Volunteers that were LFA positive were tested with an ELISA to measure IgG titers against the RBD. Subjects with titers of at least 1:1024 were selected to donate. Most donors with positive LFA had acceptable titers and were eligible to donate. Out of 171 volunteers, only 65 tested positive in the LFA (38.0%), and 55 (32.2%) had titers of at least 1:1024. Before our donation program started, 31 CP units were procured from the New York Blood Center (NYBC). Among the 31 CP units that were obtained from the NYBC, 25 units (80.6%) were positive in the LFA but only 12 units (38.7%) had titers of at least 1:1024. CP was administered to 28 hospitalized COVID-19 patients. Patients who received low titer CP, high titer CP and patients who did not receive CP were followed for 45 days after presentation. Severe adverse events were not associated with CP transfusion. Death was a less frequent outcome for patients that received high titer CP (>1:1024) 38.6% mortality, than patients that received low titer CP (≤1:1024) 77.8% mortality.

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.

Multi-drug resistant (MDR) bacteria are an increasingly serious threat to global public health. In the last decades, many studies showed that general outer membrane porins (OMPs) from Gram-negative bacteria represent the major route for antibiotics to translocate through the outer membrane.To explore the molecular rules for efficient drug transport and therefore cell killing, molecule-protein interaction is analyzed at single molecule level via electrophysiology. We incorporated OMPs into planar bilayer and single-channel currents were measured by an automated planar lipid bilayer station (Orbit 16, Nanion). The role of OMPs in the molecule-protein interactions can be characterized by the interaction kinetics. Here, we show by using 11 clinical relevant antibiotics and Omp35, Omp36 porins from Enterobacter aerogenes, that Omp35 has generally higher antibiotic-porin interaction than Omp36. Interestingly, E. aerogenes preferentially expresses Omp36 in human hosts. These in vitro results also correlate well with in vivo data collected from bacterial viability assays.On the other side, we studied the effect of magnesium on the antibiotic-porin interactions in order to approach physiological conditions. Since fluoroquinolones antibiotics have relatively heavy dipole, they can be used as probes to detect the electrostatic properties at the constriction zone of the porin channels. We found that the addition of magnesium strengthened the interaction between norfloxacin antibiotics and Omp36. The results indicate that the electrostatic properties of the constriction zone are altered and show a more efficient route to transport the norfloxacin.In conclusion, the porin properties can be better understood when considering the host environment. Improved understanding of antibiotic-porin interaction kinetics and the correlated antibiotic translocation based cell killing would facilitate the understanding on porin specificity thus the design of new antibiotics that can more efficiently reach high intracellular concentration.

The role of major porin OmpPst1 of Providencia stuartii in antibiotic susceptibility for two carbapenems is investigated by combining high-resolution conductance measurements, liposome swelling, and microbiological assays. Reconstitution of a single OmpPst1 into a planar lipid bilayer and measuring the ion current, in the presence of imipenem, revealed a concentration-dependent decrease in conductance, whereas meropenem produced well-resolved short ion current blockages. Liposome swelling assays suggested a small flux of imipenem in contrast to a rapid permeation of meropenem. The lower antibiotic susceptibility of P. stuartii to imipenem compared to meropenem correlated well with the decreased level of permeation of the former through the OmpPst1 channel.

Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.

The role of the outer-membrane channel from a mycolic acid containing Gram-positive bacteria Nocardia farcinica, which forms a hydrophilic pathway across the cell wall, was characterized. Single channel electrophysiology measurements and liposome swelling assays revealed the permeation of hydrophilic solutes including sugars, amino acids and antibiotics. The cation selective N. farcinica channel exhibited strong interaction with the positively charged antibiotics; amikacin and kanamycin, and surprisingly also with the negatively charged ertapenem. Voltage dependent kinetics of amikacin and kanamycin interactions were studied to distinguish binding from translocation. Moreover, the importance of charged residues inside the channel was investigated using mutational studies that revealed rate limiting interactions during the permeation.

Penetration through the outer cell wall is the first step for an antibiotic to reach the target site inside the bacteria. In this study, the role of major porin- OmpPst1 of Providencia stuartii is investigated in its susceptibility for two carbapenem antibiotics imipenem and meropenem. Electrophysiology measurements revealed kinetic parameters of antibiotic binding to the channel. Activity of antibiotics against bacterium determined by microbiological assays correlates with the results obtained from liposome swelling assay and planar lipid bilayer measurements. In addition, the effect of divalent and trivalent cations on antibiotic affinity to the channel is investigated. To follow the exact translocation pathway, molecular modeling provides atomistic details on the interaction of specific side chains of the two antibiotics with the channel residues and the position of affinity sites. Further, mutation of these specific sites in the channel and recording ion current fluctuation in presence of antibiotics reveal the rate limiting interaction for its translocation. Here, we have concluded on the relation between permeation of the antibiotics and resistance acquired by Providencia stuartii. Moreover, employing a multidisciplinary approach from MD simulations to protein engineering helps to get a molecular picture. This information further might give insights for rational drug design, for effective uptake of antibiotics through porins- check point of the cell.References:1. Bajaj H, et al. (2012) Biochemistry, 51, 10244-9.2. Tran QT, et al. (2010) J Biol Chem, 285, 32273-81.

The emergence and dissemination of coordinated resistance mechanism (MDR) underline the importance to understand the rate limiting steps in antibiotic action. Here OmpF porin from E. coli is used as an example to demonstrate what limits the penetration of carbapenems. Bacteriological kinetic killing assays, temperature dependent ion conductance measurements, and all-atom computer simulations were combined to study interaction and translocation of clinically relevant β-lactam antibiotics through wild type OmpF and two mutants D113A and D121A, where the key residues at the constriction region have been substituted. Expression of these various OmpF mutants in an otherwise porin-null bacterial strain revealed an increase of bacterial susceptibility for the mutants. High-resolution conductance measurements and modulating the temperature indicates lower energy barriers for mutant porin correlating with the microbiological assays. All atom modeling provided a most probable pathway able to identify the relevant side-chain interactions. This combined approach allows identifying rate limiting interaction and suggests possible modification to enhance antibiotic penetration.References:1. Hajjar E, et al. Bridging timescales and length scales: from macroscopic flux to molecular mechanism of antibiotic diffusion through porins. Biophys J. 2010, 98, 569-75.2. Singh PR, et al. Antibiotic permeation across the OmpF channel: modulation of the affinity site in the presence of magnesium. J Phys Chem B. 2012, 116, 4433-8.

The outer membrane of Gram-negative bacteria such as Escherichia coli acts as a selective permeable barrier between cell and external environment. Water filled outer membrane proteins called as porins were identified for exchange of hydrophilic solutes and hydrophilic antibiotics. One of the most abundant outer membrane porins in E. coli is OmpC and many studies revealed that down-regulation or mutation of this porin shows reduced accumulation of antibacterials in bacterial cells [1]. Fluoroquinolones, used since 1980, are the most common treatment for urinary tract infection caused by E. coli and today this treatment is ineffective in more than half of the patients globally due to widespread resistance. So far the influx kinetics of fluoroquinolones with OmpC has been characterized on free standing lipid bilayers formed on a glass substrate [2]. In particular, detailed analysis of antibiotic interaction with a single OmpC channel using electrophysiology can provide a kinetic description. Here we have investigated two fluoroquinolones, Ciprofloxacin and Enrofloxacin, using an advanced molecular dynamics technique, i.e., metadynamics [3,4]. These free energy calculations help to identify the most favorable paths and activation energies required for molecules to translocate through the OmpC channel. Furthermore, we have also investigated the translocation of the same molecules in the presence of different salts to understand the altered translocation kinetics [5]. Moreover, the identification of favorable interactions networks is important to determine the most prominent residues required for translocation.[1] H. Lou et al., PloS one 6, e25825 (2011).[2] K. R. Mahendran et al., J. Biomol. Screen. 15, 302 (2010).[3] A. Kumar et al., J. Phys.: Condens. Matter 22, 454125 (2010).[4] T. Mach et al., J. Am. Chem. Soc. 130, 13301 (2008).[5] S. Kojima et al., J. Biol. Chem. 289, 26464 (2014).