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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.

A critical step in intracellular Chlamydia infection is the production of infectious progeny through the expression of late genes. This differentiation step involves conversion from a reticulate body (RB), which is the replicating form of the bacterium, into an elementary body (EB), which is the developmental form that spreads the infection to a new host cell. EUO is an important chlamydial transcription factor that controls the expression of late genes, but the mechanisms that regulate EUO are not known. We report that a plasmid-encoded protein, Pgp4, enhanced the repressor activity of EUO. Pgp4 did not function as a transcription factor because it did not bind or directly modulate transcription of its target promoters. Instead, Pgp4 increased the ability of EUO to bind and repress EUO-regulated promoters in vitro and physically interacted with EUO in pulldown assays with recombinant proteins. We detected earlier onset of EUO-dependent late gene expression by immunofluorescence microscopy in Pgp4-deficient C. trachomatis and C. muridarum strains. In addition, the absence of Pgp4 led to earlier onset of RB-to-EB conversion in C. muridarum. These data support a role for Pgp4 as a negative regulator of chlamydial transcription that delays late gene expression. Our studies revealed that Pgp4 also has an EUO-independent function as a positive regulator of chlamydial transcription. IMPORTANCEChlamydia trachomatis is an important human pathogen that causes more than 150 million active cases of genital and eye infection in the world. This obligate intracellular bacterium produces infectious progeny within an infected human cell through the expression of late chlamydial genes. We showed that the ability of a key chlamydial transcription factor, EUO, to repress late genes was enhanced by a plasmid-encoded protein, Pgp4. In addition, studies with Chlamydia Pgp4-deficient strains provide evidence that Pgp4 delays late gene expression in infected cells. Thus, Pgp4 is a novel regulator of late gene expression in Chlamydia through its ability to enhance the repressor function of EUO.

A critical step in intracellular Chlamydia infection is the production of infectious progeny through the expression of late genes. This differentiation step involves conversion from a reticulate body (RB), which is the replicating form of the bacterium, into an elementary body (EB), which is the developmental form that spreads the infection to a new host cell. EUO is an important chlamydial transcription factor that controls the expression of late genes, but the mechanisms that regulate EUO are not known. We report that a plasmid-encoded protein, Pgp4, enhanced the repressor activity of EUO. Pgp4 did not function as a transcription factor because it did not bind or directly modulate transcription of its target promoters. Instead, Pgp4 increased the ability of EUO to bind and repress EUO-regulated promoters in vitro and physically interacted with EUO in pulldown assays with recombinant proteins. We detected earlier onset of EUO-dependent late gene expression by immunofluorescence microscopy in Pgp4-deficient C. trachomatis and C. muridarum strains. In addition, the absence of Pgp4 led to earlier onset of RB-to-EB conversion in C. muridarum. These data support a role for Pgp4 as a negative regulator of chlamydial transcription that delays late gene expression. Our studies revealed that Pgp4 also has an EUO-independent function as a positive regulator of chlamydial transcription. IMPORTANCE Chlamydia trachomatis is an important human pathogen that causes more than 150 million active cases of genital and eye infection in the world. This obligate intracellular bacterium produces infectious progeny within an infected human cell through the expression of late chlamydial genes. We showed that the ability of a key chlamydial transcription factor, EUO, to repress late genes was enhanced by a plasmid-encoded protein, Pgp4. In addition, studies with Chlamydia Pgp4-deficient strains provide evidence that Pgp4 delays late gene expression in infected cells. Thus, Pgp4 is a novel regulator of late gene expression in Chlamydia through its ability to enhance the repressor function of EUO.

The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in Brucella abortus is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of ybeY in B. abortus led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the Δ ybeY strain was significantly attenuated in both macrophage and mouse models of infection. The Δ ybeY strain also showed increased sensitivities to several in vitro -applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the Δ ybeY strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of Brucella ; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria. IMPORTANCE Brucella spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat Brucella infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in B. abortus . Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.

Metal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.