Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU).

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).

Extracellular hydrolytic potential drives microbiome shifts during anaerobic co-digestion of sewage sludge and food waste.

Bacterial community structure and dynamics in anaerobic digesters are primarily influenced by feedstock composition. It is therefore important to unveil microbial traits that explain microbiome variations in response to substrate changes. Here, gene and genome-centric metagenomics were used to examine microbiome dynamics in four laboratory-scale reactors, in which sewage sludge was co-digested with increasing amounts of food waste. A co-occurrence network revealed microbiome shifts in response to changes in substrate composition and concentration. Food waste concentration correlated with extracellular enzymes and metagenome-assembled genomes (MAGs) involved in the degradation of complex carbohydrates commonly found in fruits and plant cell walls as well as with the abundance of hydrolytic MAGs. A key role was attributed to Proteiniphillum for being the only bacteria that encoded the complete pectin degradation pathway. These results suggest that changes of feedstock composition establish new microbial niches for bacteria with the capacity to degrade newly added substrates.

Microbiome network analysis of co-occurrence patterns in anaerobic co-digestion of sewage sludge and food waste.

Addition of food waste (FW) as a co-substrate in anaerobic digesters of wastewater treatment plants is a desirable strategy towards achievement of the potential of wastewater treatment plants to become energy-neutral, diverting at the same time organic waste from landfills. Because substrate type is a driver of variations in phylogenetic structure of digester microbiomes, it is critical to understand how microbial communities respond to changes in substrate composition and concentration. In this work, high throughput sequencing was used to monitor the dynamics of microbiome changes in four parallel laboratory-scale anaerobic digesters treating sewage sludge during acclimation to an increasing amount of food waste. A co-occurrence network was constructed using data from 49 metagenomes sampled over the 161 days of the digesters' operation. More than half of the nodes in the network were clustered in two major modules, i.e. groups of highly interconnected taxa that had much fewer connections with taxa outside the group. The dynamics of co-occurrence networks evidenced shifts that occurred within microbial communities due to the addition of food waste in the co-digestion process. A diverse and reproducible group of hydrolytic and fermentative bacteria, syntrophic bacteria and methanogenic archaea appeared to grow in a concerted fashion to allow stable performance of anaerobic co-digestion at high FW.

Identification of the Acinetobacter baumannii Ribonuclease P Catalytic Subunit: Cleavage of a Target mRNA in the Presence of an External Guide Sequence.

The bacterial ribonuclease P or RNase P holoenzyme is usually composed of a catalytic RNA subunit, M1, and a cofactor protein, C5. This enzyme was first identified for its role in maturation of tRNAs by endonucleolytic cleavage of the pre-tRNA. The RNase P endonucleolytic activity is characterized by having structural but not sequence substrate requirements. This property led to development of EGS technology, which consists of utilizing a short antisense oligonucleotide that when forming a duplex with a target RNA induces its cleavage by RNase P. This technology is being explored for designing therapies that interfere with expression of genes, in the case of bacterial infections EGS technology could be applied to target essential, virulence, or antibiotic resistant genes. Acinetobacter baumannii is a problematic pathogen that is commonly resistant to multiple antibiotics, and EGS technology could be utilized to design alternative therapies. To better understand the A. baumannii RNase P we first identified and characterized the catalytic subunit. We identified a gene coding for an RNA species, M1Ab, with the expected features of the RNase P M1 subunit. A recombinant clone coding for M1Ab complemented the M1 thermosensitive mutant Escherichia coli BL21(DE3) T7A49, which upon transformation was able to grow at the non-permissive temperature. M1Ab showed in vitro catalytic activity in combination with the C5 protein cofactor from E. coli as well as with that from A. baumannii, which was identified, cloned and partially purified. M1Ab was also able to cleave a target mRNA in the presence of an EGS with efficiency comparable to that of the E. coli M1, suggesting that EGS technology could be a viable option for designing therapeutic alternatives to treat multiresistant A. baumannii infections.

Assessment of External Guide Sequences' (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules.

RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5'-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.

External guide sequence technology: a path to development of novel antimicrobial therapeutics.

RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.