Lysozyme Counteracts ß-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

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.

L-Form Switching in Escherichia coli as a Common ß-Lactam Resistance Mechanism.

Cell wall deficient bacterial L-forms are induced by exposure to cell wall-targeting antibiotics and immune effectors such as lysozyme. L-forms of different bacteria (including Escherichia coli) have been reported in human infections, but whether this is a normal adaptive strategy or simply an artifact of antibiotic treatment in certain bacterial species remains unclear. Here we show that members of a representative, diverse set of pathogenic E. coli readily proliferate as L-forms in supratherapeutic concentrations of the broad-spectrum antibiotic meropenem. We report that they are completely resistant to antibiotics targeting any penicillin-binding proteins in this state, including PBP1A/1B, PBP2, PBP3, PBP4, and PBP5/6. Importantly, we observed that reversion to the cell-walled state occurs efficiently, less than 20 h after antibiotic cessation, with few or no changes in DNA sequence. We defined for the first time a logarithmic L-form growth phase with a doubling time of 80 to 190 min, followed by a stationary phase in late cultures. We further demonstrated that L-forms are metabolically active and remain normally susceptible to antibiotics that affect DNA torsion and ribosomal function. Our findings provide insights into the biology of L-forms and help us understand the risk of ß-lactam failure in persistent infections in which L-forms may be common. IMPORTANCE Bacterial L-forms require specialized culture techniques and are neither widely reported nor well understood in human infections. To date, most of the studies have been conducted on Gram-positive and stable L-form bacteria, which usually require mutagenesis or long-term passages for their generation. Here, using an adapted osmoprotective growth media, we provide evidence that pathogenic E. coli can efficiently switch to L-forms and back to a cell-walled state, proliferating aerobically in supratherapeutic concentrations of antibiotics targeting cell walls with few or no changes in their DNA sequences. Our work demonstrates that L-form switching is an effective adaptive strategy in stressful environments and can be expected to limit the efficacy of ß-lactam for many important infections.

Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities.

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the ß-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.

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.

Isolation of L-form Bacteria from Urine using Filtration Method.

Transition of bacteria to the L-form state is thought to play a possible role in immune evasion and bacterial persistence during treatment with cell wall-targeting antibiotics. However, isolation and handling of L-form bacteria is challenging, mainly due to their high sensitivity to changes in osmolarity. Here, we describe detailed protocols for the preparation of L-form medium, isolation of L-forms from urine using a filtration method, detection of L-forms in urine samples by phase contrast microscopy and induction of L-forms in vitro. The exact requirements for survival and growth of L-forms may vary from strain to strain. Therefore, the methods presented here are intended to act as basic guidelines for establishing L-form protocols within individual laboratories, rather than as precise instructions. The filtration method can lead to a reduction in the number of L-forms in a sample and should not be used for quantification. However, it is the only method used so far for effective separation of cell wall-deficient variants from their walled counterparts and for identification of bacterial strains, which are capable of L-form switching in patients with urinary tract infections. The filtration method has the potential to be adapted for the isolation of L-forms from patients with other categories of bacterial infections and from environmental samples.

Crucial role for central carbon metabolism in the bacterial L-form switch and killing by ß-lactam antibiotics.

The peptidoglycan cell wall is an essential structure for the growth of most bacteria. However, many are capable of switching into a wall-deficient L-form state in which they are resistant to antibiotics that target cell wall synthesis under osmoprotective conditions, including host environments. L-form cells may have an important role in chronic or recurrent infections. The cellular pathways involved in switching to and from the L-form state remain poorly understood. This work shows that the lack of a cell wall, or blocking its synthesis with ß-lactam antibiotics, results in an increased flux through glycolysis. This leads to the production of reactive oxygen species from the respiratory chain, which prevents L-form growth. Compensating for the metabolic imbalance by slowing down glycolysis, activating gluconeogenesis or depleting oxygen enables L-form growth in Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus. These effects do not occur in Enterococcus faecium, which lacks the respiratory chain pathway. Our results collectively show that when cell wall synthesis is blocked under aerobic and glycolytic conditions, perturbation of cellular metabolism causes cell death. We provide a mechanistic framework for many anecdotal descriptions of the optimal conditions for L-form growth and non-lytic killing by ß-lactam antibiotics.

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.

L-form bacteria, chronic diseases and the origins of life.

The peptidoglycan cell wall is widely conserved across the bacterial domain, suggesting that it appeared early in the evolution of bacteria. It is normally essential but under certain conditions wall-deficient or 'L-form' bacteria can be isolated. In Bacillus subtilis this normally requires two genetic changes. The first, exemplified by mutations shutting down wall precursor synthesis, works by increasing membrane synthesis. This promotes the unusual form of proliferation used by L-forms, involving a range of relatively disorganized membrane blebbing or vesiculation events. The secondary class of mutations probably work by relieving oxidative stress that L-forms may incur due to their unbalanced metabolism. Repression or inhibition of cell wall precursor synthesis can stimulate the L-form transition in a wide range of bacteria, of both Gram-positive and -negative lineages. L-forms are completely resistant to most antibiotics working specifically on cell wall synthesis, such as penicillins and cephalosporins, consistent with the many reports of their involvement in various chronic diseases. They are potentially important in biotechnology, because lack of a wall can be advantageous in a range of production or strain improvement applications. Finally, L-forms provide an interesting model system for studying early steps in the evolution of cellular life.This article is part of the themed issue 'The new bacteriology'.

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.