Rli51 Attenuates Transcription of the Listeria Pathogenicity Island 1 Gene mpl and Functions as a Trans-Acting sRNA in Intracellular Bacteria.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Genomic insights from Paraclostridium bifermentans HD0315_2: General features and pathogenic potential.

Background: Paraclostridium bifermentans is the most diverse distributed species of Paraclostridium and can cause fatal human infections under rare conditions. However, its pathogenic mechanisms and adaptation ability behind infections remain unclear. Herein, we reported the complete genome sequence of P. bifermentans HD0315_2 isolated from the feces of a patient with Crohn's disease. Then, we performed genomic analyses to understand its pathogenic mechanisms and adaptation ability. Results: The de novo assembly revealed that the HD0315_2 strain carried a circular chromosome of 3.27 Mb and six circular plasmids (19.41 to 139.50 kb). The phylogenomic analysis assigned the HD0315_2 strain as P. bifermentans and reclassified some previously non-P. bifermentans strains into this clade. The general genomic features showed that this species harbored a flexible genomic pool characterized by variable genome length and multiple plasmids. Then, the HD0315_2 strain was predicted as a human pathogen with high probability, and Listeria LIPI-1 virulence proteins were identified on its genome. Besides, abundant antibiotics/metal/stress resistant genes, such as asrABCH, cat, mccF, macB, entS, albA, bcrA, and tetB, were carried by either the genome or the plasmids. Furthermore, we proposed that transposase-directed horizontal gene transfer was responsible for the distribution of multiple copies of the hin gene in the plasmids. Conclusion: The flexible genomic pool of P. bifermentans encodes abundant functions for antimicrobial or oxidative stress resistance, helping it successfully inhabit and adapt to diverse environments. Moreover, P. bifermentans HD0315_2 might infect hosts via a Listeria LIPI-1-like cycle, with the help of a plasmid expressing the Hin DNA invertase to evade host immune responses.

Comparative Genomics of Listeria monocytogenes Isolates from Ruminant Listeriosis Cases in the Midwest United States.

Ruminants are a well-known reservoir for Listeria monocytogenes. In addition to asymptomatic carriage of the pathogen, ruminants can also acquire listeriosis and develop clinical manifestations in the form of neurologic or fetal infections, similar to those occurring in humans. Genomic characterization of ruminant listeriosis cases in Europe have identified lineage 1 and 2 strains associated with infection, as well as clonal complexes (CCs) that are commonly isolated from human cases of listeriosis; however, there is little information on the diversity of L. monocytogenes from ruminant listeriosis in the United States. In this study, we characterized and compared 73 L. monocytogenes isolates from ruminant listeriosis cases from the Midwest and the Upper Great Plains collected from 2015 to 2020. Using whole-genome sequence data, we classified the isolates and identified key virulence factors, stress-associated genes, and mobile genetic elements within our data set. Our isolates belonged to three different lineages: 31% to lineage 1, 53% to lineage 2, and 15% to lineage 3. Lineage 1 and 3 isolates were associated with neurologic infections, while lineage 2 showed a greater frequency of fetal infections. Additionally, the presence of mobile elements, virulence-associated genes, and stress and antimicrobial resistance genes was evaluated. These genetic elements are responsible for most of the subgroup-specific features and may play a key role in the spread of hypervirulent clones, including the spread of hypervirulent CC1 clone commonly associated with disease in humans, and may explain the increased frequency of certain clones in the area. IMPORTANCE Listeria monocytogenes affects humans and animals, causing encephalitis, septicemia, and abortions, among other clinical outcomes. Ruminants such as cattle, goats, and sheep are the main carriers contributing to the maintenance and dispersal of this pathogen in the farm environment. Contamination of food products from farms is of concern not only because many L. monocytogenes genotypes found there are associated with human listeriosis but also as a cause of significant economic losses when livestock and food products are affected. Ruminant listeriosis has been characterized extensively in Europe; however, there is limited information about the genetic diversity of these cases in the United States. Identification of subgroups with a greater ability to spread may facilitate surveillance and management of listeriosis and contribute to a better understanding of the genome diversity of this pathogen, providing insights into the molecular epidemiology of ruminant listeriosis in the region.

Comparative genomics analyses revealed two virulent Listeria monocytogenes strains isolated from ready-to-eat food.

BACKGROUND: Listeria monocytogenes is an important foodborne pathogen that causes considerable morbidity in humans with high mortality rates. In this study, we have sequenced the genomes and performed comparative genomics analyses on two strains, LM115 and LM41, isolated from ready-to-eat food in Malaysia. RESULTS: The genome size of LM115 and LM41 was 2,959,041 and 2,963,111 bp, respectively. These two strains shared approximately 90% homologous genes. Comparative genomics and phylogenomic analyses revealed that LM115 and LM41 were more closely related to the reference strains F2365 and EGD-e, respectively. Our virulence profiling indicated a total of 31 virulence genes shared by both analysed strains. These shared genes included those that encode for internalins and L. monocytogenes pathogenicity island 1 (LIPI-1). Both the Malaysian L. monocytogenes strains also harboured several genes associated with stress tolerance to counter the adverse conditions. Seven antibiotic and efflux pump related genes which may confer resistance against lincomycin, erythromycin, fosfomycin, quinolone, tetracycline, and penicillin, and macrolides were identified in the genomes of both strains. CONCLUSIONS: Whole genome sequencing and comparative genomics analyses revealed two virulent L. monocytogenes strains isolated from ready-to-eat foods in Malaysia. The identification of strains with pathogenic, persistent, and antibiotic resistant potentials from minimally processed food warrant close attention from both healthcare and food industry.

Genome Sequence of Bacillus cereus FORC_021, a Food-Borne Pathogen Isolated from a Knife at a Sashimi Restaurant.

Bacillus cereus causes food-borne illness through contaminated foods; therefore, its pathogenicity and genome sequences have been analyzed in several studies. We sequenced and analyzed B. cereus strain FORC_021 isolated from a sashimi restaurant. The genome sequence consists of 5,373,294 bp with 35.36% GC contents, 5,350 predicted CDSs, 42 rRNA genes, and 107 tRNA genes. Based on in silico DNA-DNA hybridization values, B. cereus ATCC 14579T was closest to FORC_021 among the complete genome-sequenced strains. Three major enterotoxins were detected in FORC_021. Comparative genomic analysis of FORC_021 with ATCC 14579T revealed that FORC_021 harbored an additional genomic region encoding virulence factors, such as putative ADP-ribosylating toxin, spore germination protein, internalin, and sortase. Furthermore, in vitro cytotoxicity testing showed that FORC_021 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. This genomic information of FORC_021 will help us to understand its pathogenesis and assist in managing food contamination.