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The gatekeeper of Yersinia type III secretion is under RNA thermometer control

['Stephan Pienkoß', 'Microbial Biology', 'Ruhr University Bochum', 'Bochum', 'Soheila Javadi', 'Paweena Chaoprasid', 'Institute Of Infectiology', 'Center For Molecular Biology Of Inflammation', 'Zmbe', 'University Of Münster']

Date: 2021-11

Temperature and host cell contact (simulated by calcium depletion in vitro) certainly play a dominant role in T3SS gene expression, but several other environmental parameters influence this process and typically converge on lcrF expression. The [2Fe-2S] transcription factor IscR mediates oxygen and iron regulation of the T3SS [ 62 ]. It is thought to repress T3SS expression in the intestinal lumen and to induce T3SS expression in deeper tissues according to the gradual changes in oxygen tension and iron availability. A role of the CpxAR two-component system [ 63 ] and the Rcs phosphorelay system [ 64 ], which monitor the bacterial cell envelope integrity, in LcrF induction indicates that various other environmental cues are fed into T3SS regulation. It seems that Yersinia has coopted an array of regulatory pathways to sense and integrate the overwhelming signal complexity after transition from the outside to the mammalian gastrointestinal tract in order to precisely adjust the synthesis and activity of the costly and potentially deleterious T3SS to the ambient conditions.

Another important signal in Yersinia T3SS gene expression is Ca 2+ , a phenomenon known as low calcium response [ 60 ]. The interplay between YopN and its partner proteins ( Fig 1 ) is responsible for the calcium-controlled secretion of Yops [ 61 ]. We observed a combined effect of temperature and low calcium, which mimics the situation in the mammalian host, on yopN mRNA ( Fig 2B ) and on YopN protein even in the context of a foreign promoter ( Fig 7C ) suggesting that both transcription and translation are positively affected by low calcium by mechanisms not yet understood. Host cell contact-dependent transcriptional regulation of the T3SS and effector proteins involves a number of factors apart from LcrF, including the carbon storage regulator CsrA, the small RNAs CsrB and CsrC and YopD [ 33 , 55 ]. It is possible that this multifactorial network also has an impact on translation efficiency of the yopN transcript.

At ambient temperatures (25°C), Yersinia downregulates virulence-associated pathways while the flagellar synthesis is induced [ 56 ]. The global regulator YmoA represses the expression of the main virulence regulator lcrF at 25°C [ 53 , 57 – 59 ]. Besides, RNA thermometers (RNATs) also contribute to repression of specific genes like lcrF itself or the secretion regulator yopN [ 34 ]. At virulence-relevant temperatures (37°C), YmoA is degraded by proteases which leads to the derepression of lcrF [ 59 ]. Furthermore, melting of the lcrF RNAT increases the expression induction and synthesis of the virulence regulator and thus induce the expression of T3SS genes and effector protein genes (Yop genes) [ 34 ]. The transcript of yopN posseses an RNAT that additionally induces its expression at 37°C. In calcium-containing environments, YopN together with TyeA and the chaperone complex SycN/YscB, prevents the secretion of Yops by blocking the T3SS channel in the cytosol. In contrast, the complex dissociates under calcium deficiency allowing first the secretion of YopN and subsequently the secretion of further Yops into the surrounding medium [ 20 , 22 , 23 , 26 , 27 ]. Blue box: closed RNAT; red box: open RNAT; red circles: Yops; blue circles: YopN.

Since the entire T3SS is encoded on a 70-kbp virulence plasmid, the copy number of this plasmid matters. An immediate strategy of Yersinia to boost the expression of T3SS genes under adequate conditions is by increasing the plasmid copy number [ 52 ]. Gene dose elevation is followed by a multi-faceted regulation of virulence gene expression. Ambient temperature plays a key role in this process. For intestinal pathogens such as Y. pseudotuberculosis, 37°C is a reliable indicator of successful invasion of a mammalian host. The expression of more than 300 genes changes at least four-fold between cultures grown at 25 or 37°C indicating a major temperature-dependent reprogramming of bacterial metabolism and physiology [ 32 ]. Temperature-responsive virulence gene expression centers around lcrF coding for the primary virulence transcription factor of the Ysc-T3SS/Yop machinery ( Fig 9 ). Transcription of the yscW-lcrF operon at low temperatures outside the host is partially repressed by the global histone-like regulator YmoA. In addition, translation of residual transcripts is inhibited by an RNAT in the intergenic region between yscW and lcrF [ 53 , 54 , 34 ]. Three mechanisms contribute to the induction of LcrF levels at 37°C: (i) a temperature-dependent topology change of the yscW-lcrF promoter, (ii) the proteolytic cleavage of YmoA by the Lon and ClpP proteases, and (iii) melting of the lcrF RNAT [ 34 , 52 – 54 ]. To make LcrF-mediated virulence gene induction not solely dependent on the temperature signal, the pathogen has installed additional mechanisms that control the concentration of the transcription factor. Signals indicating host cell contact are integrated via the translocon protein YopD, which–when not secreted–ultimately stimulates lcrF mRNA degradation by the degradosome [ 55 ].

Many bacterial pathogens use a T3SS as effective syringe-type device to inject effector proteins into eukaryotic cells. The biosynthesis of the individual components, the correct assembly and dynamic exchange of subunits comes at a considerable cost to the bacterium. Once fully assembled, traffic through the T3SS must be controlled because the unrestricted translocation of effector proteins in the absence of host contact leads to a severe growth arrest ( Fig 7 ). Growth inhibition might also be due to the loss of essential ions and amino acids through the open T3SS [ 48 – 50 ]. It is therefore not at all surprising that T3S is a tightly regulated process that responds to environmental and host cues with many checks and balances [ 51 ].

A novel RNAT at a critical checkpoint of T3SS

The contribution of our study to understanding the intricate control of Yersinia virulence lies in the discovery of yet another layer of temperature regulation of the Yersinia T3SS. Unlike LcrF-mediated control, this mechanism does not concern the expression of the entire secretion machinery but addresses a highly specific process, the gating of the secretion channel. We identified essentially the same RNAT in both biological isoforms of the yopN 5’-UTR, of which the short one (37 nts) is much more abundant than the longer one (102 nts). A functional RNAT in both a long and a short UTR also exists upstream of Y. pseudouberculosis cnfY [41]. Roughly equal activity of the RNAT in the short and long yopN transcripts can be explained by an identical arrangement of the structure that sequesters the translation initiation region.

The architecture of the short yopN RNAT is rather simple and composed of only a single hairpin (Fig 2B). This makes it one of the shortest known natural RNATs. Many other RNATs contain several hairpins upstream of the decisive thermolabile structure. The adjoining, often more stable hairpins are believed to aid in proper folding of the weaker regulatory hairpin [35]. RNAT-containing 5’-UTRs with a length below 50 nts are exceptional. One example of such a simple helix with an internal asymmetric loop was found upstream of Synechocystis hsp17 [37,65]. Sequence-wise, the Yersinia yopN thermometer is unique and bears no resemblance to RNATs in Yersinia or other bacteria. This is contrasted by the lcrF RNAT, which belongs to the fourU thermometer family, in which the SD sequence is paired by four uridines [34,66]. Structure-wise, the yopN thermometer follows the most common principle. The SD region folds back onto an upstream region that has already been transcribed and is waiting for interaction. While this might have considerable advantages in co-transcriptional RNA folding and instantaneous repression of translation, there are exceptions to this rule. For example, the SD sequence of the Neisseria meningitidis fHbp transcript coding for the factor H binding protein folds onto a downstream sequence in the coding region [67].

The necessary temperature-sensitivity in the yopN thermosensor is built in by three mismatches opposite the SD sequence. Partial stabilization of this internal loop by a central CG pair was not sufficient to eliminate temperature responsiveness (R2 in Fig 3). Closing the entire loop by three nucleotides complementary to the SD sequence impaired melting and translation of the yopN mRNA at host body temperature (R1 in Figs 3–6) and resulted in massive leakage of Yops accompanied by growth cessation (Fig 7). In line with other reports [34,40,68–71], these results show how the manipulation of just a few nucleotides in the non-coding region of a protein-coding transcript can dramatically influence expression and the corresponding biological outcome. The results also reinforce the concept that RNAT function depends on a delicate balance of stabilizing and destabilizing elements that render the RNA structure responsive to temperature fluctuations within a narrow and physiologically permissive temperature range. Previous NMR studies provided in-depth insights into the critical contribution of destabilizing internal bulges and loops and non-Watson-Crick-type base pairs to the functional design of various heat shock and virulence thermometers [65,72–74].

The position of the yopN RNAT at the beginning of the multicistronic virA mRNA suggests that the six downstream genes of the operon are unaffected by this translational control element. RNAT-mediated differential regulation of the first or second gene of a bistronic operon has been reported for the Salmonella groESL operon and Yersinia yscW-lcrF operon, respectively [34,75]. RNATs or other riboregulators, such as riboswitches or small regulatory RNAs, provide a simple means to differentially control individual genes in complex operon arrangements [76].

Why is it important to have yopN expression under such strict regulation? The conservation of the 5’-UTR sequence in various Yersinia species strongly supports its functional relevance. Otherwise, this sequence outside the coding region would be free to evolve. The absence of RNAT-like elements upstream of the virB and virC operons (S3 Fig) lends further support to the importance of the conserved RNAT upstream of yopN. It is conceivable that the additional checkpoint imposed by the RNAT slightly delays the synthesis of YopN in comparison to other T3SS proteins that are all under the regulatory umbrella of LcrF. Interestingly, the virA operon is less dependent on LcrF than the other vir operons [77]. In the sequential assembly of the T3SS it might be counterproductive to have the gatekeeper present when the T3SS is not ready yet. Despite its limited size, YopN has at least four protein interaction partners, namely TyeA, SycN and YscB (Fig 1), and YscI, a component of the inner rod of the T3SS [78]. The appropriate order of intermolecular interactions between these proteins might require the precise adjustment of the cellular concentration of the individual components. Another possibility is that YopN has presently undisclosed functions that need to be kept in check when the conditions are not appropriate. Recent studies suggest that YopN has functions beyond its role as gatekeeper of the T3SS. The centrally located coiled-coil domains of YopN encompassing amino acids 65–100 provides a virulence-related function. The region contributes to the translocation of YopE and YopH, and is required for systemic infection in mice [29,30].

Another scenario, in which the yopN RNAT might play a role, is the return to lower temperatures after shedding from the host to the environment. The reversible nature of zipper-like RNATs allows them to respond to both increasing and decreasing temperatures. Adjusting translation to the new situation and preventing the overabundance of YopN might be another reason for the presence of an RNAT.

Our data suggests that YopN is an active rather than passive gatekeeper that exerts a directional influence on Yop secretion. The absence of YopN triggered an uncontrolled premature burst of Yops into the medium, which prevented efficient targeting into host cells. Reduced YopE-TEM translocation by the ΔyopN strain with or without the R1 variant was associated with less rounded epithelial cells compared to infections with the WT strain and the D2 variant. Delayed rounding of HeLa cells infected with the ΔyopN strain and a delayed cytotoxic response has previously been observed [46,79] and let to a model, in which YopN promotes host cell contact [80]. In combination with the finding that YopN is involved in the secretion of YopH and YopE [29,30], these results suggest that YopN regulates the secretion hierarchy to accomplish an ordered translocation of Yops into host cells. Precise timing of the initiation and termination of T3S is critical to efficiently interfere with deleterious immune cell function such as phagocytosis after secretion. While several facets of the biological role of YopN remain unexplored, the cumulative results of the present and other studies suggest functions of this versatile protein that are worth further exploration. Since the incorrect intracellular concentration of YopN causes severe phenotypes, any strategy interfering with the provision of this protein might be suited to combat the pathogenic outcome of Yersinia infections.

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