The Escherichia coli DH5α and Y. pestis strains were routinely grown in heart infusion broth (HIB) or on tryptose blood agar (TBA) base plates (Difco, Detroit, MI) at 27°C (Y. pestis) or 37°C (E. coli). Appropriate antibiotics were added to culture media when needed with ampicillin (50 μg/ml), kanamycin (50 μg/ml), chloramphenicol (20 μg/ml), and streptomycin (50 μg/ml). Non-polar deletion of Y. pestis KIM5 chromosomal DNA sequences ylrC (y3397; codons 29-515), ylrB (y3399; codons 29-261), and ylrA (y3400; codons 23-529) was accomplished using lambda Red recombination as described by Datsenko and Wanner (2000). PCR products used to construct the gene replacement were amplified using the template plasmid pKD4 (Km^r). The resulting PCR products were gel purified, ethanol precipitated, and resuspended in 10 μl of distilled water. Y. pestis KIM5 strain carrying plasmid pKD46, which encodes the Red recombinase, was induced with 0.2% L-arabinose for 2 h prior to harvest. Electrocompetent cells were electroporated with the purified PCR products. The transformations were plated onto TBA plates containing kanamycin (50 μg/ml). Plasmid pCP20, which encodes the FLP recombinase, was electroporated into the Km^r resistant strains to facilitate the removal of the FLP recognition target-flanked kan cassette and plasmid pKD46 simultaneously. Plasmid pCP20 was cured from the Y. pestis deletion strains by overnight growth at 39°C. Additionally, a deletion strain of the three gene sequences ylrC-codon 29 to ylrA-codon 529 was constructed using lambda Red-recombination as described above.

A TraSH-based approach was used to map and quantify the relative abundance of the different transposon mutants in order to identify transposon mutant insertion sites that are underrepresented in the population of mutants exposed to RAW264.7 murine macrophage-like cells. The details of the mutagenesis, infection, and TraSH-screen and data analysis are described in Bartra et al. (2012).

Cell infection assays were performed essentially as described by Rosenzweig et al. (2005). RAW 264.7 murine macrophage-like cells and HeLa cells were cultured in Dubelcco's modified Eagle's medium (DMEM, Gibco) containing 10% heat-inactivated fetal bovine serum (Cellgro) at 37°C in the presence of 5% CO₂. Cells were seeded in 24-well plates at densities of 2.5–4.0 × 10⁵ per well. Bacterial cell cultures were grown in tissue culture (TC) medium overnight at 27°C, diluted into fresh TC media and incubated with shaking at 27°C for 2 h and then shifted to 37°C for 1 h prior to being added to the cultured macrophages at a multiplicity of infection (MOI) of 30. After a 30 min attachment period, fresh DMEM medium was added to each well. At the 0 h and 8 h time points, the infected cells were lysed with 500 μl of distilled H₂O and a portion of which was plated on TBA plates. Colony forming units (CFU) were counted after 2–3 days. For mouse infections, all procedures were in strict accordance with federal and state government guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and their use was approved for this entire study by the University of Miami institutional animal care and use committee (protocol number 15-081). Mice were infected intravenously with a total of 2000 CFU (1000 CFU each of the parental Y. pestis KIM5 and the isogenic ΔyopB or ΔylrABC strains). Mice were humanely sacrificed at 48 h post infection, spleens were removed and homogenized in sterile water containing 0.05% triton X-100 by grinding through a fine wire mesh. The resulting homogenates were diluted and plated on media containing either chloramphenicol (CM) to select for the CM-resistant parental strain, as well as antibiotic-free media that allowed growth of both the parental strain and the CM-sensitive mutant strains. Two to three days later colonies were enumerated and the competition index (CI) for the parental/ ΔyopB and parental/ΔylrABC co-infected animals was computed by dividing the CFU of the mutant by the CFU of the parental strain.

DNA fragments used encoding YlrA, YlrB, and YlrC were PCR amplified from chromosomal DNA of Y. pestis KIM5. The resultant DNA fragments were digested with HindIII and BglII and inserted into HindIII- and BglII-digested pFLAG-CTC vector (Sigma-Aldrich). These vectors express full-length C-terminal FLAG-tagged YlrA-FLAG, YlrB-FLAG, and YlrC-FLAG. In addition, DNA sequences predicted to encode the YlrA, YlrB, and YlrC N-terminal T3S signal (SS) (amino acid residues 2 to 10) were deleted from each expression vector using whole plasmid PCR (Imai et al., 1991), generating plasmids pYlrA-FLAG-ΔSS, pYlrB-FLAG-ΔSS, and pYlrC-FLAG-ΔSS. Oligonucleotide pairs used were YlrASS-F and YlrASS-R, YlrBSS-F and YlrBSS-R, and YlrCSS-F and YlrCSS-R The resultant Ylr expression plasmids were transformed into Y. pestis KIM8Δ4 (Bartra et al., 2006).

Expression plasmids encoding full length YlrA, YlrB, and YlrC carrying a C-terminal β-lactamase gene were constructed by the PCR-ligation-PCR technique (Ali and Steinkasserer, 1995). Individual Y. pestis KIM genes and upstream sequences that include each gene's ribosomal binding site were amplified by PCR from vectors encoding full-length FLAG-YlrA, FLAG-YlrB, and FLAG-YlrC, respectively, using oligonucleotides primer pairs YlrA-KpnI-F and YlrA-FL-R, YlrB-KpnI-F and YlrB-FL-R, and YlrC-KpnI-F and YlrC-FL-R. The DNA fragments encoding the Bla gene were amplified from plasmid pBSKII- using primers Bla-25-F and Bla-STOP-HindIII-R. Obtained DNA fragments were gel purified, kinased and ligated. The reaction was used for a second PCR using primers YlrA-KpnI-F and Bla-STOP-HindIII-R, YlrB-KpnI-F and Bla-STOP-HindIII-R, and YlrC-KpnI-F and Bla-STOP-HindIII-R. The resulting DNA fragments were ethanol precipitated, digested with KpnI and HindIII, and inserted into KpnI and HindIII-digested pBad₁₈-Cm^r. The resultant plasmids were transformed into a Y. pestis strain lacking YopE, YopJ, YopM, YopT, YopH, and YpkA.

DNA fragments encoding YlrA, YlrB and YlrC were amplified by PCR from pFLAG-YlrA, pFLAG-YlrB, and pFLAG-YlrC, respectively, using oligonucleotides primer pairs YlrA-XhoI and YlrA-BamHI, YlrB-XhoI and YlrB-BamHI, and YlrC-XhoI and YlrC-BamHI listed in Table 2. The resultant DNA fragments were digested with BamHI and XhoI and inserted into BamHI- and XhoI-digested pREP3X. The pREP-3X vector contains the inducible nmt1 promoter (Maundrell, 1993) and the presence of thiamine (15 μM) in the medium represses the promoter. If thiamine is not present in the media, the promoter becomes activated resulting in the transcription of the ylr gene cloned downstream. Plasmids generated were pREP-3X-YlrA, pREP-3X-YlrB, and pREP-3X-YlrC. In addition, site-specific mutation of three catalytic residues of the putative NEL domain of YlrA was done using the whole plasmid PCR method (Imai et al., 1991) generating plasmids pREP-3X-YlrA-C386S, pREP-3X-YlrA-R389A, and pREP-3X-YlrA-S449F. Schizosaccharomyces pombe h⁻ ade6-704 leu1-32 ura4-D18 was grown in Pombe Glutamate medium (PMG) at 32°C. The plasmid vector pREP3x (nmt13x Thiamine repressible promoter) was used to express the Ylr genes in fission yeast. The plasmid vectors were transformed using the lithium acetate protocol (Morita and Takegawa, 2004). The yeast strains were grown in PMG supplemented with the appropriate amino acids as well as thiamine to maintain selection for 2 days in logarithmic growth to an optical density (OD) of 0.1–0.4 at 595 nm. After growth, the cells were centrifuged at 4,000 × g, washed 3 times with distilled sterile water, and diluted for an overnight growth in media lacking thiamine. The following day, the OD of the cultures at 595 nm was measured and the cultures were diluted to an OD of 0.1. Three 10-fold serial dilutions of the cultures were made, and 5 μL of each dilution was spotted onto an agar plate with and without thiamine and grown at 32°C for 3 days.

For standard secretion assays, Y. pestis strains were grown in thoroughly modified Higuchi's (TMH) medium in the presence or absence of 2.5 mM CaCl₂ for 1 h at 27°C and then shifted to 37°C for the next 5 h as previously described (Jackson et al., 1998). FLAG-tagged Ylr proteins (pFLAG-CTC vector) were induced with 0.05 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at the temperature shift.

Cultures of bacteria were harvested by centrifugation at 14,000 × g for 10 min at room temperature. Pellets of whole-cell bacteria and trichloroacetic acid (TCA)-precipitated supernatant proteins were resuspended according to the harvest OD₆₂₀ and subjected to SDS-PAGE and immunoblotting as previously described (Jackson et al., 1998). LcrV was detected with rabbit polyclonal antisera (1:20,000) raised against the full-length LcrV proteins. FLAG-tagged proteins were detected with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) (1:1,000).

HeLa cells were seeded to a six-well plate to achieve a 40–50% confluence 1 day prior to infection. Y. pestis strains were grown at 27°C for 2 h in HIB with antibiotics as appropriate and then shifted to 37°C for 1 h to induce expression of T3SS proteins. At temperature shift, 0.2% L-arabinose was added to the cultures. Bacteria were added at MOI = 50 to HeLa cells. The infected cells were incubated at 37°C with 5% CO₂. Two negative controls (without Y. pestis cells and Y. pestis with pBAD18-pYscF₁-Bla) and a positive control were used to determine the background blue and green fluorescence. After infection for 3 h, the growth medium from the cells was removed and the cells were washed with 500 μL of 1x of GIBCO Hank's Balanced Salt Solution (HBSS). 6X CCF-AM (Invitrogen) was added to each sample to obtain a final concentration of 1X per manufacturer's instructions. The plate was covered and incubated in room temperature for 1 h. Cells were then visualized by confocal-fluorescent microscopy (Leica TCS SP5 Inverted Confocal Microscope).

Previously we described a transposon site hybridization (TraSH)-based screen to identify Y. pestis loci that are required during infection of cultured mouse macrophage-like RAW 264.7 cells (Bartra et al., 2012). From a pool of approximately 90,000 unique variants, 44 Y. pestis ORFs were identified that appeared to be required for pathogen survival during its interaction with the macrophage. A number of these ORFs (17) are encoded on the extrachromosomal plasmids pCD1 and pPCP1 and express components of the T3SS and Pla protease that both play critical roles in Y. pestis virulence (Viboud and Bliska, 2005). One such pCD1-encoded locus among this set of 17 identified ORFs, YopM, possesses Leucine-Rich Repeats (LRRs), and is associated with a variety of infection phenotypes (see Introduction). Among the 27 chromosomally-encoded pro-survival ORFs, there were three, y3397, y3399, and y3400, that were previously predicted to possess LRRs (Hu et al., 2016) and here will be designated as Yersinia leucine-rich repeat A (ylrA), B (ylrB), and C (ylrC), respectively (Figure 1). In addition to LRR-encoding sequences, these three ORFs also possess predicted secretion signals (SS) for the T3SS at their N-termini and YlrA and YlrC are additionally predicted to encode novel ubiquitin E3 ligase (NEL) domains (Wang et al., 2013; Marchler-Bauer et al., 2017).

The aforementioned TraSH screen involves infecting macrophages with a mixture of Y. pestis variants followed by a quantification of under-represented genetic loci. To test whether the Ylr-encoding loci play a pro-survival function for Y. pestis during a single-strain infection, individual ΔylrA, ΔylrB, and ΔylrC mutant strains were constructed and tested in a colony-forming unit (CFU)-based infection assay. Similar to what has been shown previously, the fold-increases of the parental Y. pestis KIM5 strain and the T3SS mutant stain (ΔyopB) during a 8 h infection, were 170 and 3, respectively (Figure 2A; Rosenzweig et al., 2005). The infectivity of the single gene deletion ΔylrA, ΔylrB, and ΔylrC mutant strains, as well as the triple gene deletion ΔylrABC mutant strain were intermediate between the parental and T3SS mutant strains (Figure 2A). This intermediate in vitro infection phenotype is similar to that observed in Yersinia mutants individually deleted for the YopE and YopH T3SS effectors (Bartra et al., 2001). These data validated the findings of the TraSH screen indicating that the Ylr-encoding genes promote the survival of Y. pestis during their interaction with macrophages.

The Y. pestis ΔylrABC strain was tested in a mouse-based infection model to determine whether the Ylr-encoding loci promote the pathogen survival in vivo. A competition infection assay was used to compare the proliferation of the parental KIM5 strain to that of the mutant strain within individual mice. Accordingly, in the control experiment mice were infected with an equal mixture of the parental KIM5 and the isogenic T3SS mutant ΔyopB strains and after 2 days the mice were humanely sacrificed and the presence of each strain in the spleen was determined by CFU assay. In contrast to the equal ratio of the parental and ΔyopB stains in the “input” (i.e., the dosed inoculum), the parental strain was greatly over-represented in the “output” (i.e., the splenic homogenates derived from mice infected for 2 days) (Figure 2B). Mice similarly infected with an equal mixture of the parental KIM5 and the isogenic ΔylrABC strains also yielded a statistically significant over-representation of the parental strain following 2 days of infection (Figure 2B). There was no detectable differences in growth rates between the parental KIM5 and the isogenic ΔylrABC strains (Supplementary Figure 1). Collectively these data indicate that the ylr locus promotes Y. pestis infection both in cells and in mice.

Sequence analysis of the ylrA, ylrB, and ylrC genes indicated that they each potentially encode at their amino-terminus a recognition sequence for the type III secretion system (T3SS) (Hu et al., 2016). To determine if the YlrA, YlrB, and YlrC proteins are secreted by the T3SS, expression vectors containing full-length C-terminal FLAG-tagged proteins were constructed. In addition, DNA sequences predicted to encode YlrA, YlrB, and YlrC N-terminal T3SS signal residues 2 to 10 were deleted from each of the expression vectors. Generated plasmids were placed in Y. pestis KIM8Δ4, a strain containing a modified pCD1 virulence plasmid that contains all of the genes necessary to assemble a functional T3SS but lacks all other effector proteins (Bartra et al., 2006). The strains were tested for expression and secretion in the presence and absence of 2.5 mM of calcium and bacterial pellet and supernatant were analyzed. FLAG-tagged YlrA, YlrB, and YlrC were expressed in the presence and absence of calcium, but were secreted only in the absence of calcium (Figure 3; red boxes; and Data Sheet 1). There was no secretion of the YlrA, YlrB, and YlrC proteins when the putative T3SS signal of each protein was deleted. Finally, there was no secretion of YlrA-FLAG, YlrB-FLAG, or YlrC-FLAG detected by Y. pestis strain KIM8 (pCD1-) or Y. pestis ΔyscF, both of which are unable to assemble a functional Ysc injectisome (Supplementary Figures 2–4). Together, these results demonstrate that YlrA, YlrB and YlrC are T3S substrates and that they are recognized and secreted by the Y. pestis T3SS.

A β-lactamase reporter system was used to determine whether Ylr proteins are translocated into cultured cells during infection (Marketon et al., 2005). Upon diffusion into cultured cells, CCF2-AM is cleaved into the membrane-impermeable CCF2 by endogenous cytoplasmic esterases, emitting a green fluorescence. When β-lactamase fusion proteins are injected into the cells, CCF2 can be cleaved further, changing the fluorescent emission to blue (Charpentier and Oswald, 2004). Cells that were either uninfected or infected with a Y. pestis strain expressing YscF₁-Bla, which is expressed but not secreted by the T3SS, retained their initial green fluorescence (Figure 4). In striking contrast, essentially all CCF2-labeled cells infected with a Y. pestis strain expressing YopM-Bla, which, as discussed in the Introduction, is a plasmid-encoded virulence factor, fluoresce blue indicating that YopM-Bla is readily translocated into the infected cell cytosol. Cells infected with Y. pestis strains expressing either YlrA-Bla or YlrC-Bla retained their green fluorescence indicating that these proteins were not detectably translocated as assayed by this method despite being readily expressed as assayed by western blotting (Figure 4; Supplementary Figure 5). In contrast, blue fluorescing cells were readily detected following infection with a Y. pestis strain expressing YlrB-Bla. These show that the YlrB is translocated by Y. pestis during infection.

Previous studies of the Yersinia Yop virulence factors have shown a robust correlation between their growth inhibition in yeast and their activity in mammalian cells (Lesser and Miller, 2001; Wiley et al., 2009). Yeast provide a relatively simple genetic model system for the study of the eukaryotic genes and is based on the premise that yeast share many molecular, genetic, and biochemical features with mammalian cells (Zhao and Lieberman, 1995). Accordingly, the fission yeast Schizosaccharomyces pombe was transformed with either an empty vector control plasmid or inducible expression plasmids encoding either YlrA, YlrB, or YlrC and plated either on media in which the Ylr proteins were not expressed (“uninduced”) or on media in which Ylr expression occurs (“induced”). All S. pombe transformant strains grew equally well on uninduced media (Figure 5). However, on induced media, there was a marked differences between the relatively rapid growth of the empty vector and YlrB transformants (rows 1 and 6) and that of the relatively poorer growth of the YlrA and YlrC transformants (rows 2 and 7). Previous work has shown that IpaH effectors and SspH2 have a conserved catalytic cysteine residue that is absolutely required for ubiquitin ligase activity (Rohde et al., 2007; Quezada et al., 2009). To determine if this residue and other conserved residues of the NEL domain of YlrA will affect its growth-inhibiting activity in yeast, these residues were substituted generating plasmids expressing YlrA-C386S, YlrA-R389A, and YlrA-S449F. There was a clear reduction in growth inhibition for the S. pombe strain expressing the YlrA-C386S variant compared to the strain expressing wild-type YlrA (Figure 5, rows 2 and 3). In contrast, there was no detectable loss of growth inhibitory activity in the S. pombe strains expressing either the YlrA-R389A or YlrA-S449F (rows 4 and 5). These data suggest that the ubiquitin ligase activity of YlrA is required for its growth inhibitory activity in eukaryotic cells.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.