Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

A comprehensive review of small regulatory RNAs in Brucella spp.

Brucella spp. are Gram-negative bacteria that naturally infect a variety of domesticated and wild animals, often resulting in abortions and sterility. Humans exposed to these animals or animal products can also develop debilitating, flu-like disease. The brucellae are intracellular pathogens that reside predominantly within immune cells, typically macrophages, where they replicate in a specialized compartment. This capacity of Brucella to survive and replicate within macrophages is essential to their ability to cause disease. In recent years, several groups have identified and characterized small regulatory RNAs (sRNAs) as critical factors in the control of Brucella physiology within macrophages and overall disease virulence. sRNAs are generally < 300 nucleotides in length, and these independent sRNA transcripts are encoded either next to (i.e., cis-encoded) or at a distant location to (i.e., trans-encoded) the genes that they regulate. Trans-encoded sRNAs interact with the mRNA transcripts through short stretches of imperfect base pairing that often require the RNA chaperone Hfq to facilitate sRNA-mRNA interaction. In many instances, these sRNA-mRNA interactions inhibit gene expression, usually by occluding the ribosome-binding site (RBS) and/or by decreasing the stability of the mRNA, leading to degradation of the transcript. A number of sRNAs have been predicted and authenticated in Brucella strains, and a variety of approaches, techniques, and means of validation have been employed in these efforts. Nonetheless, some important issues and considerations regarding the study of sRNA regulation in Brucella need to be addressed. For example, the lack of uniform sRNA nomenclature in Brucella has led to difficulty in comparisons of sRNAs across the different Brucella species, and there exist multiple names in the literature for what are functionally the same sRNA. Moreover, even though bona fide sRNAs have been discovered in Brucella, scant functional information is known about the regulatory activities of these sRNAs, or the extent to which these sRNAs are required for the intracellular life and/or host colonization by the brucellae. Therefore, this review summarizes the historical context of Hfq and sRNAs in Brucella; our current understanding of Brucella sRNAs; and some future perspectives and considerations for the field of sRNA biology in the brucellae.

ASC-Mediated Inflammation and Pyroptosis Attenuates Brucella abortus Pathogenesis Following the Recognition of gDNA.

Brucella abortus is a zoonotic pathogen that causes brucellosis. Because of Brucella's unique LPS layer and intracellular localization predominately within macrophages, it can often evade immune detection. However, pattern recognition receptors are capable of sensing Brucella pathogen-associated molecular patterns (PAMPS). For example, NOD-like receptors (NLRs) can form a multi-protein inflammasome complex to attenuate Brucella pathogenesis. The inflammasome activates IL-1ß and IL-18 to drive immune cell recruitment. Alternatively, inflammasome activation also initiates inflammatory cell death, termed pyroptosis, which augments bacteria clearance. In this report, we assess canonical and non-canonical inflammasome activation following B. abortus infection. We conducted in vivo studies using Asc-/- mice and observed decreased mouse survival, immune cell recruitment, and increased bacteria load. We also conducted studies with Caspase-11-/- mice and did not observe any significant impact on B. abortus pathogenesis. Through mechanistic studies using Asc-/- macrophages, our data suggests that the protective role of ASC may result from the induction of pyroptosis through a gasdermin D-dependent mechanism in macrophages. Additionally, we show that the recognition of Brucella is facilitated by sensing the PAMP gDNA rather than the less immunogenic LPS. Together, these results refine our understanding of the role that inflammasome activation and pyroptosis plays during brucellosis.

The Endoribonuclease RNase E Coordinates Expression of mRNAs and Small Regulatory RNAs and Is Critical for the Virulence of Brucella abortus.

RNases are key regulatory components in prokaryotes, responsible for the degradation and maturation of specific RNA molecules at precise times. Specifically, RNases allow cells to cope with changes in their environment through rapid alteration of gene expression. To date, few RNases have been characterized in the mammalian pathogen Brucella abortus In the present work, we sought to investigate several RNases in B. abortus and determine what role, if any, they have in pathogenesis. Of the 4 RNases reported in this study, the highly conserved endoribonuclease, RNase E, was found to play an integral role in the virulence of B. abortus Although rne, which encodes RNase E, is essential in B. abortus, we were able to generate a strain encoding a defective version of RNase E lacking the C-terminal portion of the protein, and this strain (rne-tnc) was attenuated in a mouse model of Brucella infection. RNA-sequencing analysis revealed massive RNA dysregulation in B. abortusrne-tnc, with 122 upregulated and 161 downregulated transcripts compared to the parental strain. Interestingly, several mRNAs related to metal homeostasis were significantly decreased in the rne-tnc strain. We also identified a small regulatory RNA (sRNA), called Bsr4, that exhibited significantly elevated levels in rne-tnc, demonstrating an important role for RNase E in sRNA-mediated regulatory pathways in Brucella Overall, these data highlight the importance of RNase E in B. abortus, including the role of RNase E in properly controlling mRNA levels and contributing to virulence in an animal model of infection.IMPORTANCE Brucellosis is a debilitating disease of humans and animals globally, and there is currently no vaccine to combat human infection by Brucella spp. Moreover, effective antibiotic treatment in humans is extremely difficult and can lead to disease relapse. Therefore, it is imperative that systems and pathways be identified and characterized in the brucellae so new vaccines and therapies can be generated. In this study, we describe the impact of the endoribonuclease RNase E on the control of mRNA and small regulatory RNA (sRNA) levels in B. abortus, as well as the importance of RNase E for the full virulence of B. abortus This work greatly enhances our understanding of ribonucleases in the biology and pathogenesis of Brucella spp.

The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus aureus.

Small RNAs (sRNAs) remain an understudied class of regulatory molecules in bacteria in general and in Gram-positive bacteria in particular. In the major human pathogen Staphylococcus aureus, hundreds of sRNAs have been identified; however, only a few have been characterized in detail. In this study, we investigate the role of the sRNA Teg41 in S. aureus virulence. We demonstrate that Teg41, an sRNA divergently transcribed from the locus that encodes the cytolytic alpha phenol-soluble modulin (αPSM) peptides, plays a critical role in αPSM production. Overproduction of Teg41 leads to an increase in αPSM levels and a corresponding increase in hemolytic activity from S. aureus cells and cell-free culture supernatants. To identify regions of Teg41 important for its function, we performed an in silico RNA-RNA interaction analysis which predicted an interaction between the 3' end of Teg41 and the αPSM transcript. Deleting a 24-nucleotide region from the S. aureus genome, corresponding to the 3' end of Teg41, led to a 10-fold reduction in αPSM-dependent hemolytic activity and attenuation of virulence in a murine abscess model of infection. Restoration of hemolytic activity in the Teg41Δ3' strain was possible by expressing full-length Teg41 in trans Restoration of hemolytic activity was also possible by expressing the 3' end of Teg41, suggesting that this region of Teg41 is necessary and sufficient for αPSM-dependent hemolysis. Our results show that Teg41 is positively influencing αPSM production, demonstrating for the first time regulation of the αPSM peptides by an sRNA in S. aureusIMPORTANCE The alpha phenol-soluble modulins (αPSMs) are among the most potent toxins produced by Staphylococcus aureus Their biological role during infection has been studied in detail; however, the way they are produced by the bacterial cell is not well understood. In this work, we identify a small RNA molecule called Teg41 that plays an important role in αPSM production by S. aureus Teg41 positively influences αPSM production. The importance of Teg41 is highlighted by the fact that a strain containing a deletion in the 3' end of Teg41 produces significantly less αPSMs and is attenuated for virulence in a mouse abscess model of infection. As the search for new therapeutic strategies to combat S. aureus infection proceeds, Teg41 may represent a novel target.