Examination of Xenorhabdus nematophila lipases in pathogenic and mutualistic host interactions reveals a role for xlpA in nematode progeny production

Abstract

Xenorhabdus nematophila is a gammaproteobacterium and broad-host-range insect pathogen. It is also a symbiont of Steinernema carpocapsae, the nematode vector that transports the bacterium between insect hosts. X. nematophila produces several secreted enzymes, including hemolysins, lipases, and proteases, which are thought to contribute to virulence or nutrient acquisition for the bacterium and its nematode host in vivo. X. nematophila has two lipase activities with distinct in vitro specificities for Tween and lecithin. The gene encoding the Tween-specific lipase, xlpA, has been identified and is not required for X. nematophila virulence in one insect host, the tobacco hornworm Manduca sexta. However, the gene encoding the lecithin-specific lipase activity is not currently known. Here, we identify X. nematophila estA, a gene encoding a putative lecithinase, and show that an estA mutant lacks in vitro lipase activity against lecithin but has wild-type virulence in Manduca sexta. X. nematophila secondary-form phenotypic variants have higher in vitro lecithinase activity and estA transcript levels than do primary-form variants, and estA transcription is negatively regulated by NilR, a repressor of nematode colonization factors. We establish a role for xlpA, but not estA, in supporting production of nematode progeny during growth in Galleria mellonella insects. Future research is aimed at characterizing the biological roles of estA and xlpA in other insect hosts.

FIG. 1.

The estA (A) and xlpA (B) loci of X. nematophila. Arrows indicate genes and their directions of transcription. The regions of estA predicted to encode a phospholipase/lecithinase/hemolysin domain and an autotransporter domain are designated by white and gray shadings, respectively. Genes were named based on their similarity to those of E. coli, with the exceptions of estA and xlpA, which were named based on their homologs in S. liquefaciens and Yersinia enterocolitica, respectively.

FIG. 2.

Host interactions of X. nematophila lipase mutants. Ability of logarithmic-phase (A) or stationary-phase (B) X. nematophila cultures to kill M. sexta insects (note that virulence of logarithmic-phase xlpA2::Km cultures are from a study by Richards et al. [42] and are included for comparison). Cultures were injected, and percent mortality at 72 h is shown. (C) X. nematophila colonization of S. carpocapsae nematodes cultivated on lawns of each strain. The wild type (black bars; wt) and estA1::Km (white bars), xlpA2::Km (cross-hatched bars), and estA1::Km xlpA3::Sm (diagonal lines) mutants are shown. Separate wild-type results are presented because independent experiments were performed for each mutant. Error bars represent standard errors (n = 3). No significant differences were found (P > 0.05).

FIG. 3.

Progeny production of nematodes colonized by X. nematophila lipase mutants. (A) IJ nematodes colonized by X. nematophila wild type (black bars; wt) or lrhA2 (vertical lines) estA1::Km (white bars), xlpA2::Km (cross-hatched bars), or estA1::Km xlpA3::Sm (diagonal lines) mutants were injected into G. mellonella larvae, and the time to first emergence of progeny IJs was recorded. (B) Larvae were then monitored for total progeny IJ emergence at the indicated days. Nematodes with wild-type (black squares, solid lines) or lrhA2 (white circles), estA1::Km (white diamonds), xlpA2::Km (white squares), or estA1::Km xlpA3::Str (white triangles) mutant treatment are shown. Different letters indicate significantly different values (P < 0.05; n = 10). In the case of panel B, significant differences refer to cumulative numbers at day 28.

FIG. 4.

Transcript levels of the X. nematophila estA gene. Total cellular RNA was extracted from logarithmic-phase X. nematophila cultures of primary-form wild type (black bar), secondary-form wild type (white bar), and the estA1::Km mutant (vertical lines, not visible) (A) or wild-type (black bar), nilR16::Sm (cross-hatched bar), and nilR16::Sm carrying a wild-type copy of nilR (diagonal lines) (B). cDNA was analyzed by qPCR. Levels of transcript are reported as percentages of primary-form wild type. Bars with different letters are significantly different from each other (P < 0.05; n ≥ 3).