The Small Regulatory RNAs LhrC1–5 Contribute to the Response of Listeria monocytogenes to Heme Toxicity

Abstract

The LhrC family of small regulatory RNAs (sRNAs) is known to be induced when the foodborne pathogen Listeria monocytogenes is exposed to infection-relevant conditions, such as human blood. Here we demonstrate that excess heme, the core component of hemoglobin in blood, leads to a strong induction of the LhrC family members LhrC1–5. The heme-dependent activation of lhrC1–5 relies on the response regulator LisR, which is known to play a role in virulence and stress tolerance. Importantly, our studies revealed that LhrC1–5 and LisR contribute to the adaptation of L. monocytogenes to excess heme. Regarding the regulatory function of the sRNAs, we demonstrate that LhrC1–5 act to down-regulate the expression of known LhrC target genes under heme-rich conditions: oppA, tcsA, and lapB, encoding surface exposed proteins with virulence functions. These genes were originally identified as targets for LhrC-mediated control under cell envelope stress conditions, suggesting a link between the response to heme toxicity and cell envelope stress in L. monocytogenes. We also investigated the role of LhrC1–5 in controlling the expression of genes involved in heme uptake and utilization: lmo2186 and lmo2185, encoding the heme-binding proteins Hbp1 and Hbp2, respectively, and lmo0484, encoding a heme oxygenase-like protein. Using in vitro binding assays, we demonstrated that the LhrC family member LhrC4 interacts with mRNAs encoded from lmo2186, lmo2185, and lmo0484. For lmo0484, we furthermore show that LhrC4 uses a CU-rich loop for basepairing to the AG-rich Shine–Dalgarno region of the mRNA. The presence of a link between the response to heme toxicity and cell envelope stress was further underlined by the observation that LhrC1–5 down-regulate the expression of lmo0484 in response to the cell wall-acting antibiotic cefuroxime. Collectively, this study suggests a role for the LisR-regulated sRNAs LhrC1–5 in a coordinated response to excess heme and cell envelope stress in L. monocytogenes.

Role and regulation of the stress activated sigma factor sigma B (σB) in the saprophytic and host-associated life stages of Listeria monocytogenes

“…(Mollerup et al, 2016). In contrast to the other six LhrC sibling sRNAs, the expression of Rli33-1 is not induced by heme toxicity (Dos Santos et al, 2018). Alongside LhrC1-5, the expression of Rli33-1 is required for L. monocytogenes infection of macrophages (Mraheil et al, 2011).…”

The LhrC sRNAs control expression of T cell-stimulating antigen TcsA in Listeria monocytogenes by decreasing tcsA mRNA stability

“…As whole human blood contains components integral to the host's immune response, and surface-exposed proteins are readily recognized by the immune system, the LhrCmediated down-regulation of OppA, LapB, and TcsA might represent a mechanism by which L. monocytogenes evades host immunity [12]. More recently, we have shown that LhrC1-5 are induced in response to excess heme, the core component of hemoglobin in blood [25]. We demonstrated that LhrC1-5 contribute to the adaptation of L. monocytogenes to excess heme, and that LhrC1-5 down-regulate the expression of genes involved in heme uptake and utilization, including lmo2186, lmo2185 (which encode the heme-binding proteins Hbp1 and Hbp2, respectively) and lmo0484 (which encodes a heme oxygenase-like protein) [25].…”

“…More recently, we have shown that LhrC1-5 are induced in response to excess heme, the core component of hemoglobin in blood [25]. We demonstrated that LhrC1-5 contribute to the adaptation of L. monocytogenes to excess heme, and that LhrC1-5 down-regulate the expression of genes involved in heme uptake and utilization, including lmo2186, lmo2185 (which encode the heme-binding proteins Hbp1 and Hbp2, respectively) and lmo0484 (which encodes a heme oxygenase-like protein) [25]. As previously demonstrated for oppA and lapB, we found that LhrC4 interacts with lmo2186, lmo2185, and lmo0484 mRNAs and, for lmo0484, LhrC4 uses a CU-rich loop for base-pairing to the AG-rich RBS of the mRNA [25].…”

“…For northern blot and primer extension assays, total RNA was extracted as previously described [25]. Briefly, bacterial cells were harvested by centrifugation for 3 min at 5000g at 4°C and snap-frozen in liquid nitrogen.…”

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

“…Rli33-1 sRNA belongs to the multicopy family LhrC, encompassing 7 members [ 77 ]. Besides Rli33-1, LhrC sRNAs, comprising LhrC1-5 and Rli22, are positively regulated by the TCS LisRK that responds to cell envelope stress [ 71 , 72 , 76 , 77 ]. Transcription of all LhrC sRNAs except Rli22 is strongly induced during intracellular bacterial growth in murine macrophages.…”