Mycoplasma gallisepticum (MG) mainly infects chickens to initiate chronic respiratory disease (CRD). microRNAs (miRNAs) play vital roles according to previously reported studies. Our previous study showed that gga-miR-16-5p, in MG-infected lungs of chicken embryo, was upregulated by Illumina sequencing. The study aimed to reveal what role gga-miR-16-5p plays in CRD progression. gga-miR-16-5p was upregulated in MG-infected fibroblast cells (DF-1). Phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was demonstrated as the target gene of gga-miR-16-5p. Furthermore, PIK3R1 expression was lower in MG-infected groups than it in noninfected controls measured by qPCR. Additionally, overexpressed gga-miR-16-5p could downregulate PIK3R1 and phosphorylated serine/threonine kinase (p-Akt) to express protein, whereas there is an opposite effect on inhibition. Overexpressed gga-miR-16-5p resulted in decreased activity of tumor necrosis factor alpha (TNF-α) and the nuclear factor-kappaB (NF-κB) by qPCR. Furthermore, overexpressed gga-miR-16-5p restricted cell multiplication, cycle progression, and increased apoptosis of MG-infected DF-1 cells, whereas inhibited gga-miR-16-5p led to the opposite effect. Collectively, upregulated gga-miR-16-5p could decrease multiplication, cycle progression, and increase apoptosis of MG-infected DF-1 cells, at least partly through directly targeting PIK3R1 and inhibiting PI3K/Akt/NF-κB pathway to exert an anti-inflammatory effect. Our results will provide more experimental evidence to bring pathogenesis of MG infection to light.

High-mobility group box 1 (HMGB1), a member of damage-associated molecular patterns (DAMPs), is involved in the immune regulation of several infectious diseases. Mycoplasma gallisepticum (MG) infection is proved to cause an abnormal immune response, but the role of HMGB1 in MG-induced chronic respiratory disease (CRD) is unclear. In this study, we found that HMGB1 was released from the nucleus to the extracellular in macrophages upon infection with MG. Extracellular HMGB1 bound to TLR2 activating the NF-κB pathway triggering a severe inflammatory storm and promoting the progression of MG infection. More importantly, TLR4 could be activated by HMGB1 to trigger immune disorders after TLR2 was silenced. This disease process could be interrupted by ethyl pyruvate (EP) inhibition of HMGB1 release or glycyrrhizic acid (GA). Furthermore, treatment of MG-infected chickens with GA significantly alleviated immune organ damage. In conclusion, we demonstrate that HMGB1 is secreted extracellularly to form an inflammatory environment upon MG infection, triggering a further cellular inflammatory storm in a positive feedback approach. Blocking MG-induced HMGB1 release or suppression downstream of the HMGB1-TLR2/TLR4 axis may be a promising novel strategy for the treatment of CRD. Furthermore, this study may provide a theoretical reference for understanding non-LPS-activated TLR4 events.

Mycoplasma gallisepticum (MG), a pathogen that infects chickens and some other birds, triggers chronic respiratory disease (CRD) in chickens, which is characterized by inflammation. The investigation of microbial pathogenesis would contribute to the deep understanding of infection control. Since microribonucleic acids (miRNAs) play a key role in this process, gga-mir-146c, an upregulated miRNA upon MG infection, was selected according to our previous RNA-sequencing data. In this paper, we predicted and validated that MMP16 is one of gga-miR-146c target genes. Results show that MMP16 is the target of gga-miR-146c and gga-miR-146c can downregulate MMP16 expression within limits. gga-miR-146c upregulation significantly increased the expression of TLR6, NF-κB p65, MyD88, and TNF-α, whereas the gga-miR-146c inhibitor led to an opposite result. gga-miR-146c upregulation effectively decreased apoptosis and stimulated DF-1 cells proliferation upon MG infection. On the contrary, gga-miR-146c inhibitor promoted apoptosis and repressed the proliferation. Collectively, our results suggest that gga-miR-146c upregulation upon MG infection represses MMP16 expression, activating TLR6/MyD88/NF-κB pathway, promoting cell proliferation by inhibiting cell apoptosis, and, finally, enhancing cell cycle progression to defend against host MG infection.

Proteus mirabilis can transfer transposons, insertion sequences, and gene cassettes to the chromosomes of other hosts through SXT/R391 integrative and conjugative elements (ICEs), significantly increasing the possibility of antibiotic resistance gene (ARG) evolution and expanding the risk of ARGs transmission among bacteria. A total of 103 strains of P. mirabilis were isolated from 25 farms in China from 2018 to 2020. The positive detection rate of SXT/R391 ICEs was 25.2% (26/103). All SXT/R391 ICEs positive P. mirabilis exhibited a high level of overall drug resistance. Conjugation experiments showed that all 26 SXT/R391 ICEs could efficiently transfer to Escherichia coli EC600 with a frequency of 2.0 × 10 ⁻⁷ to 6.0 × 10 ⁻⁵ . The acquired ARGs, genetic structures, homology relationships, and conservation sequences of 26 (19 different subtypes) SXT/R391 ICEs were investigated by high-throughput sequencing, whole-genome typing, and phylogenetic tree construction. ICE Pmi ChnHBRJC2 carries erm (42 ), which have never been found within an SXT/R391 ICE in P. mirabilis , and ICE Pmi ChnSC1111 carries 19 ARGs, including clinically important cfr , bla _CTX-M-65 , and aac(6')-Ib-cr , making it the ICE with the most ARGs reported to date. Through genetic stability, growth curve, and competition experiments, it was found that the transconjugant of ICE Pmi ChnSCNNC12 did not have a significant fitness cost on the recipient bacterium EC600 and may have a higher risk of transmission and dissemination. Although the transconjugant of ICE Pmi ChnSCSZC20 had a relatively obvious fitness cost on EC600, long-term resistance selection pressure may improve bacterial fitness through compensatory adaptation, providing scientific evidence for risk assessment of horizontal transfer and dissemination of SXT/R391 ICEs in P. mirabilis . IMPORTANCE: The spread of antibiotic resistance genes (ARGs) is a major public health concern. The study investigated the prevalence and genetic diversity of integrative and conjugative elements (ICEs) in Proteus mirabilis , which can transfer ARGs to other hosts. The study found that all of the P. mirabilis strains carrying ICEs exhibited a high level of drug resistance and a higher risk of transmission and dissemination of ARGs. The analysis of novel multidrug-resistant ICEs highlighted the potential for the evolution and spread of novel resistance mechanisms. These findings emphasize the importance of monitoring the spread of ICEs carrying ARGs and the urgent need for effective strategies to combat antibiotic resistance. Understanding the genetic diversity and potential for transmission of ARGs among bacteria is crucial for developing targeted interventions to mitigate the threat of antibiotic resistance.

We report a catalytic single QD nanosensor for simple, selective, and highly sensitive quantification of piRNA.