Mutagenesis of Ly49B reveals key structural elements required for promiscuous binding to MHC class I molecules and new insights into the molecular evolution of Ly49s.

Ly49B is a potentially important immunoregulator expressed on mouse myeloid cells, and it is thus an unusual member of the wider Ly49 family whose members are ordinarily found on NK cells. Ly49B displays substantial sequence divergence from other Ly49s and in particular shares virtually no amino acid sequence identity with the residues that have been reported to bind to MHC class I (cI) ligands in other Ly49s. Despite this, we show in this study that the BALB/c, but not the C57, isoform of Ly49B displays promiscuous cI binding. Binding was not significantly affected by inactivation of any of the four predicted N-linked glycosylation sites of Ly49B, nor was it affected by removal of the unique 20-aa C-terminal extension found in Ly49B. However, transfer of these C-terminal 20 aa to Ly49A inhibited cI binding, as did the addition of a hemagglutinin tag to the C terminus of Ly49B, demonstrating unexpectedly that the C-terminal region of Ly49s can play a significant role in ligand binding. Systematic exchange of BALB/c and C57 residues revealed that Trp(166), Asn(167), and Cys(251) are of major importance for cI binding in Ly49B. These residues are highly conserved in the Ly49 family. Remarkably, however, Ly49B(BALB) variants that have C57 residues at positions 166 or 167, and are unable to bind cI multimers, regain substantial cI binding when amino acid changes are made at distal positions, providing an explanation of how highly divergent Ly49s that retain the ability to bind cI molecules might have evolved.

The expression and function of the NKRP1 receptor family in C57BL/6 mice.

NKRP1 receptors were discovered more than 20 years ago, but due to a lack of appropriate reagents, our understanding of them has remained limited. Using a novel panel of mAbs that specifically recognize mouse NKRP1A, D, and F molecules, we report here that NKRP1D expression is limited to a subpopulation of NK cells, but in contrast to Ly49 receptors appears to be expressed in a normal codominant manner. NKRP1D(-) and NKRP1D(+) NK cells are functionally distinct, NKRP1D(+) cells showing reduced expression of various Ly49 receptors, elevated expression of CD94/NKG2 receptors, and higher IFN-gamma secretion and cytotoxicity than NKRP1D(-) cells. Furthermore, NKRP1D(+) NK cells were unable to kill transfected cells expressing high levels of Clr-b molecules, but readily killed MHC class-I-deficient blast cells that express only low levels of Clr-b. NKRP1A and NKRP1F were expressed at low levels on all splenic and bone marrow NK cells, but mAb-induced cross-linking of NKRP1A and NKRP1F caused no significant enhancement or inhibition of NK cell cytotoxicity and no detectable production of IFN-gamma. NKRP1A, D, and F expression could not be detected on NKT cells, all of which express NKRP1C, and although some activated T cells expressed NKRP1C and perhaps low levels of NKRP1A, no significant expression of NKRP1D or F could be detected. NKRP1 molecules expressed on NK cells or transfectants were down-regulated by cross-linking with mAbs or cell surface ligands, and using this phenomenon as a functional assay for NKRP1-ligand interaction revealed that NKRP1F can recognize CLR-x.

Author Correction: Possible role of L-form switching in recurrent urinary tract infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Possible role of L-form switching in recurrent urinary tract infection.

Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E. coli strains from patient samples readily transition from the walled state to L-form during challenge with a cell wall targeting antibiotic. Following antibiotic withdrawal, they then efficiently transition back to the walled state. E. coli switches between walled and L-form states in a zebrafish larva infection model. The results suggest that L-form switching is a physiologically relevant phenomenon that may contribute to the recurrence of infection in older patients with rUTI, and potentially other infections.

Comprehensive analysis of transcript start sites in ly49 genes reveals an unexpected relationship with gene function and a lack of upstream promoters.

Comprehensive analysis of the transcription start sites of the Ly49 genes of C57BL/6 mice using the oligo-capping 5'-RACE technique revealed that the genes encoding the "missing self" inhibitory receptors, Ly49A, C, G, and I, were transcribed from multiple broad regions in exon 1, in the intron1/exon2 region, and upstream of exon -1b. Ly49E was also transcribed in this manner, and uniquely showed a transcriptional shift from exon1 to exon 2 when NK cells were activated in vitro with IL2. Remarkably, a large proportion of Ly49E transcripts was then initiated from downstream of the translational start codon. By contrast, the genes encoding Ly49B and Q in myeloid cells, the activating Ly49D and H receptors in NK cells, and Ly49F in activated T cells, were predominantly transcribed from a conserved site in a pyrimidine-rich region upstream of exon 1. An ∼200 bp fragment from upstream of the Ly49B start site displayed tissue-specific promoter activity in dendritic cell lines, but the corresponding upstream fragments from all other Ly49 genes lacked detectable tissue-specific promoter activity. In particular, none displayed any significant activity in a newly developed adult NK cell line that expressed multiple Ly49 receptors. Similarly, no promoter activity could be found in fragments upstream of intron1/exon2. Collectively, these findings reveal a previously unrecognized relationship between the pattern of transcription and the expression/function of Ly49 receptors, and indicate that transcription of the Ly49 genes expressed in lymphoid cells is achieved in a manner that does not require classical upstream promoters.