A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence

Abstract

Pasteurella multocida is the causative agent of fowl cholera in birds. In a previous study using signature-tagged mutagenesis, we identified a mutant, AL251, which was attenuated for virulence in mice and in the natural chicken host. Sequence analysis indicated that AL251 had an insertional inactivation of the gene waaQ(PM), encoding a putative heptosyl transferase, required for the addition of heptose to lipopolysaccharide (LPS) (M. Harper, J. D. Boyce, I. W. Wilkie, and B. Adler, Infect. Immun. 71:5440-5446, 2003). In the present study, using mass spectrometry and nuclear magnetic resonance, we have confirmed the identity of the enzyme encoded by waaQ(PM) as a heptosyl transferase III and demonstrated that the predominant LPS glycoforms isolated from this mutant are severely truncated. Complementation experiments demonstrated that providing a functional waaQ(PM) gene in trans can restore both the LPS to its full length and growth in mice to wild-type levels. Furthermore, we have shown that mutant AL251 is unable to cause fowl cholera in chickens and that the attenuation observed is not due to increased serum sensitivity.

FIG. 1.

Analysis of P. multocida LPS by SDS-PAGE and silver staining of whole-cell lysates. (A) Comparison of P. multocida LPS profiles from wild-type VP161 (lane 1), heptosyltransferase mutant AL251 (lane 2), control strain AL438 (AL251 containing vector plasmid pAL99) (lane 3), and the complemented mutant strain AL298 (lane 4). (B) Comparison of LPS profiles of P. multocida heptosyltransferase mutant AL251 (lane 1), wild-type VP161 (lane 2), and P. multocida wild-type revertants isolated from three different chickens inoculated with AL251 (lanes 3 to 5).

FIG. 2.

Negative-ion CE-ESI-MS of P. multocida core OS. (a) Doubly charged region of core OS from parent strain VP161; (b) singly charged region of core OS from mutant strain AL251.

FIG. 3.

Region of the ¹H-NMR spectrum of the core OS derived from the LPS of P. multocida parent strain VP161 (a) and P. multocida mutant strain AL251 (b). The spectra were recorded at 25°C and referenced against internal acetone at 2.225 ppm.

FIG. 4.

Region of the NOESY spectrum of P. multocida VP161 core OS. NOE connectivities are as indicated. (Inset), Structure of the inner-core OS from VP161. The spectrum was recorded at 25°C and referenced against internal acetone at 2.225 ppm.

FIG. 5.

Proposed structures of inner core LPS of P. multocida from parent strain VP161 (a) and mutant strain AL251 (b), where R is the OS chain extension beyond Glc. Based on negative-ion CE-MS data shown in Table 3, extension of the mutant LPS molecule to include the structures shown in parentheses occurs at only low frequency (less than 4%).