Genome-wide mutant fitness profiling identifies nutritional requirements for optimal growth of Yersinia pestis in deep tissue

Abstract

Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposon mutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injection. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fitness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complementation effects. We also identified more than 30 genes with roles in nutrient acquisition and metabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes-encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)-both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes.

Importance: Our understanding of the functions required by bacteria to grow in deep tissues is limited, in part because most growth studies of pathogenic bacteria are conducted on laboratory media that do not reflect conditions prevailing in infected animal tissues. Improving our knowledge of this aspect of bacterial biology is important as a potential pathway to the development of novel therapeutics. Yersinia pestis, the plague bacterium, is highly virulent due to its rapid dissemination and growth in deep tissues, making it a good model for discovering bacterial adaptations that promote rapid growth during infection. Using Tn-seq, a genome-wide fitness profiling technique, we identified several functions required for fitness of Y. pestis in vivo that were not previously known to be important. Most of these functions are needed to acquire or synthesize nutrients. Interference with these critical nutrient acquisition pathways may be an effective strategy for designing novel antibiotics and vaccines.

FIG 1

Dense transposon mapping of the Y. pestis genome allows identification of regions under selection. The number of insertions sequenced at each site is represented by bar height (log scale). Circular plots were generated using the software program CGView (62). (A) Transposon insertions mapping to the chromosome. Large gaps correspond to blocks of essential genes, such as the NADH dehydrogenase subunits (inset; insertions are shown by the Integrated Genomics Viewer software tool [63, 64] on a log scale). (B) Transposon insertions mapping to the type III secretion plasmid pCD1. Insertions in many genes in the first 30 kb of pCD1, which encode regulatory and structural components of the type III secretion system, are selected against in vivo (outer ring; red bars) but not in vitro (inner ring; blue bars). The gap of insertions at the 60-kb mark corresponds to plasmid replication genes.

FIG 2

Selection of chromosomal Y. pestis genes during growth in vitro on rich medium. (A) The relative fitness of mutants in each chromosomal ORF plotted against significance (calculated by resampling; see Text S1 in the supplemental material). Genes harboring no sequenced insertions in the central 90% of the ORF (relative fitness = 0) are shown in the box at the left of the plot. (B) Relative fitness (histogram) correlates well to essentiality, as predicted by the hidden Markov model recently published by DeJesus and Ioerger (36) (red, essential; yellow, growth-deficient; green, nonessential). Genes harboring no sequenced insertions in the central 90% of the ORF (relative fitness = 0) are shown at the left of the plot. (C) Three examples of genes with modest reductions in relative fitness that were predicted to be essential by the hidden Markov model. Many genes in this class contain groups of consecutive TA sites that do not harbor insertions (red boxes), suggesting domain architecture. For example, the apparently essential N terminus of the rne gene is highly homologous to the essential E. coli gene of the same name (65, 66), while the evidently nonessential C terminus is highly divergent from the E. coli homologue, as has been reported for several other species (67); also shown is the apparently essential C-terminal DNA-binding domain of the rcsB gene, a transcriptional activator with multiple roles, including small RNA production and cell division (68). Insertions are shown by the Integrated Genomics Viewer software tool (63, 64) on a log scale.

FIG 3

Comparison of Tn-seq data sets to find chromosomal genes required during mammalian infection. (A) Volcano plot for identification of chromosomal genes selected in vivo. Each gene was compared between two in vitro data sets (filled pale blue squares) or between an in vitro data set and the in vivo data set (gray circles). Genes experiencing significant selection in vivo are represented by black circles. For each gene, relative fitness of insertion mutants in vivo is given with respect to the fitness of a wild-type strain. Significance is calculated by permuting the number of insertions sequenced at each TA in the gene between the two data sets (see Text S1 in the supplemental material). Genes selected in vivo appear in the upper-left quadrant, while any genes that harbor more insertions in vivo than in vitro should track to the upper-right quadrant. Genes harboring no sequenced insertions in vivo in the central 90% of the ORF (relative fitness = 0) are shown in the box at the left of the plot. (B) Genes experiencing strong selection (psn and brnQ) harbor many fewer insertions after growth in vivo. The same effect is present but less pronounced in genes experiencing moderate levels of selection (ptsG). Insertions are shown by the Integrated Genome Viewer tool (63, 64) on a log scale.

FIG 4

Y. pestis requires the branched-chain amino acid importer BrnQ for virulence in mice. (A) Left: subcutaneous infection with 1,000 CFU KIM1001 (w.t.) (n = 9), KIM1001ΔbrnQ (ΔbrnQ) (n = 17), or the complemented strain KIM1001ΔbrnQ(pSP6) (complemented) (n = 5). Mice infected with KIM1001 or KIM1001ΔbrnQ(pSP6) died within the first week of infection, while the mutant KIM1001ΔbrnQ strain failed to sicken or kill mice. Right: previous infection with KIM1001ΔbrnQ protected 15 out of 16 mice from subcutaneous challenge with 1,000 CFU KIM1001 28- to 30 days later. (B) Growth of the attenuated strains JG150A (w.t.), JG150AΔbrnQ (ΔbrnQ), and JG150AΔbrnQ(pSP6) (complemented) in the defined serum-like medium SNM alone (left) or supplemented with 2 mM leucine, isoleucine, and valine (right). Curves are representative of two independent experiments.

FIG 5

The glucose importer encoded by ptsG has little effect on Y. pestis virulence. Subcutaneous infections with 1,000 CFU KIM1001 (w.t.) (n = 10) or KIM1001ΔptsG (ΔptsG) (n = 14) were carried out. Thirteen out of fourteen mice infected with KIM1001ΔptsG died, with kinetics similar to those of mice infected with KIM1001.

FIG 6

ptsG provides a competitive advantage in the presence of glucose. (A) A 10⁴-CFU dose of wild-type KIM1001 and KIM1001ΔptsG (mixed 1:1) was injected intravenously into BALB/C or C57BL/6 mice. The proportion of KIM1001ΔptsG mutants in the population of Y. pestis in the spleen was determined 2 and 42 h postinfection by plating on TB supplemented with 2.5 mM CaCl₂, 1% glucose, and 50 µg/ml tetrazolium red. (B) The attenuated strains JG102 and JG102ΔptsG were mixed 1:1 in the rich medium TB, in TB supplemented with 5 mM glucose, and in the defined medium SNM (n = 5 for each condition). The proportion of JG102ΔptsG in each culture was measured before and after 42 h of log-phase growth.

FIG 7

Many genes on the type III secretion plasmid pCD1 undergo strong selection during infection. Each gene on pCD1 was compared between two in vitro data sets (filled pale blue squares) or between an in vitro data set and the in vivo data set (gray circles). Genes experiencing significant selection in vivo are represented by black circles. Genes harboring no sequenced insertions in vivo in the central 90% of the ORF (relative fitness = 0) are shown in the box at the left of the plot. The effector proteins of the type III secretion system undergo variable levels of selection (red, yopH; yellow, yopE; dark blue, yopK; orange, ypkA; purple, yopM; pink, yopT; green, yopJ), while the ATPase (tan, yscN) and structural components (tan, yscF) undergo strong selection.