Computationally Designed AMPs with Antibacterial and Antibiofilm Activity against MDR Acinetobacter baumannii.

The discovery of new antimicrobials is necessary to combat multidrug-resistant (MDR) bacteria, especially those that infect wounds and form prodigious biofilms, such as Acinetobacter baumannii. Antimicrobial peptides (AMPs) are a promising class of new therapeutics against drug-resistant bacteria, including gram-negatives. Here, we utilized a computational AMP design strategy combining database filtering technology plus positional analysis to design a series of novel peptides, named HRZN, designed to be active against A. baumannii. All of the HRZN peptides we synthesized exhibited antimicrobial activity against three MDR A. baumannii strains with HRZN-15 being the most active (MIC 4 µg/mL). This peptide also inhibited and eradicated biofilm of A. baumannii strain AB5075 at 8 and 16 µg/mL, which is highly effective. HRZN-15 permeabilized and depolarized the membrane of AB5075 rapidly, as demonstrated by the killing kinetics. HRZN 13 and 14 peptides had little to no hemolysis activity against human red blood cells, whereas HRZN-15, -16, and -17 peptides demonstrated more significant hemolytic activity. HRZN-15 also demonstrated toxicity to waxworms. Further modification of HRZN-15 could result in a new peptide with an improved toxicity profile. Overall, we successfully designed a set of new AMPs that demonstrated activity against MDR A. baumannii using a computational approach.

GATR-3, a Peptide That Eradicates Preformed Biofilms of Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a gram-negative bacterium that causes hospital-acquired and opportunistic infections, resulting in pneumonia, sepsis, and severe wound infections that can be difficult to treat due to antimicrobial resistance and the formation of biofilms. There is an urgent need to develop novel antimicrobials to tackle the rapid increase in antimicrobial resistance, and antimicrobial peptides (AMPs) represent an additional class of potential agents with direct antimicrobial and/or host-defense activating activities. In this study, we present GATR-3, a synthetic, designed AMP that was modified from a cryptic peptide discovered in American alligator, as our lead peptide to target multidrug-resistant (MDR) A. baumannii. Antimicrobial susceptibility testing and antibiofilm assays were performed to assess GATR-3 against a panel of 8 MDR A. baumannii strains, including AB5075 and some clinical strains. The GATR-3 mechanism of action was determined to be via loss of membrane integrity as measured by DiSC3(5) and ethidium bromide assays. GATR-3 exhibited potent antimicrobial activity against all tested multidrug-resistant A. baumannii strains with rapid killing. Biofilms are difficult to treat and eradicate. Excitingly, GATR-3 inhibited biofilm formation and, more importantly, eradicated preformed biofilms of MDR A. baumannii AB5075, as evidenced by MBEC assays and scanning electron micrographs. GATR3 did not induce resistance in MDR A. baumannii, unlike colistin. Additionally, the toxicity of GATR-3 was evaluated using human red blood cells, HepG2 cells, and waxworms using hemolysis and MTT assays. GATR-3 demonstrated little to no cytotoxicity against HepG2 and red blood cells, even at 100 µg/mL. GATR-3 injection showed little toxicity in the waxworm model, resulting in a 90% survival rate. The therapeutic index of GATR-3 was estimated (based on the HC50/MIC against human RBCs) to be 1250. Overall, GATR-3 is a promising candidate to advance to preclinical testing to potentially treat MDR A. baumannii infections.

Ab initio Designed Antimicrobial Peptides Against Gram-Negative Bacteria.

Antimicrobial peptides (AMPs) are ubiquitous amongst living organisms and are part of the innate immune system with the ability to kill pathogens directly or indirectly by modulating the immune system. AMPs have potential as a novel therapeutic against bacteria due to their quick-acting mechanism of action that prevents bacteria from developing resistance. Additionally, there is a dire need for therapeutics with activity specifically against Gram-negative bacterial infections that are intrinsically difficult to treat, with or without acquired drug resistance. Development of new antibiotics has slowed in recent years and novel therapeutics (like AMPs) with a focus against Gram-negative bacteria are needed. We designed eight novel AMPs, termed PHNX peptides, using ab initio computational design (database filtering technology combined with the novel positional analysis on APD3 dataset of AMPs with activity against Gram-negative bacteria) and assessed their theoretical function using published machine learning algorithms, and finally, validated their activity in our laboratory. These AMPs were tested to establish their minimum inhibitory concentration (MIC) and half-maximal effective concentration (EC50) under CLSI methodology against antibiotic resistant and antibiotic susceptible Escherichia coli and Staphylococcus aureus. Laboratory-based experimental results were compared to computationally predicted activities for each of the peptides to ascertain the accuracy of the computational tools used. PHNX-1 demonstrated antibacterial activity (under high and low-salt conditions) against antibiotic resistant and susceptible strains of Gram-positive and Gram-negative bacteria and PHNX-4 to -8 demonstrated low-salt antibacterial activity only. The AMPs were then evaluated for cytotoxicity using hemolysis against human red blood cells and demonstrated some hemolysis which needs to be further evaluated. In this study, we successfully developed a design methodology to create synthetic AMPs with a narrow spectrum of activity where the PHNX AMPs demonstrated higher antibacterial activity against Gram-negative bacteria compared to Gram-positive bacteria. Thus, these peptides present novel synthetic peptides with a potential for therapeutic use. Based on our findings, we propose upfront selection of the peptide dataset for analysis, an additional step of positional analysis to add to the ab initio database filtering technology (DFT) method, and we present laboratory data on the novel, synthetically designed AMPs to validate the results of the computational approach. We aim to conduct future in vivo studies which could establish these AMPs for clinical use.

Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes.

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.