Researchers at University of California at Berkley and at Institut [sic] Pasteur and INSERM in Paris wanted to understand why some bacteria are pathogenic, while others are not. A commonality among pathogenic bacteria is the ability to escape or safely inhabit lysozyme-filled macrophage vacuoles, or to encounter lysozyme elsewhere in a host’s system without sustaining damage. In this research study, the scientists worked with Listeria monocytogenes (L. monocytogenes) to discover how pathogenicity is affected by the ability to withstand this innate host defense. They discovered that almost all of the pathogenic and nonpathogenic bacteria are programmed to produce the enzymes needed to survive exposure to lysozyme, but relatively few use that ability .
L. monocytogenes is a ubiquitous, Gram-positive, intracellular human and animal pathogen . Most infections begin with the ingestion of food contaminated with the bacteria. It is commonly found on food because refrigeration only slows its growth, and because it can survive several widely-used food processing techniques. Outbreaks have been traced to soft cheeses, deli meat, unpasteurized milk, jellied pork, undercooked poultry, and coleslaw. A sample of grocery store foods showed those items most likely to be contaminated include: seafood, soft cheeses and meat products .
Assessment of disease symptoms and recent patient history gives insight into possible causes, but a definitive diagnosis in listeriosis is made only by identification of a culture through polymerase chain reaction (PCR) of that culture’s DNA. Patient samples can come from bloodwork, cerebral spinal fluid, placental blood and other normally sterile environments in the body . Non-invasive listeriosis infects the gastrointestinal tract, but invasive listeriosis is much more serious because it can spread past the walls of the digestive system, potentially causing death . The average incubation period is approximately three weeks from exposure to the onset of clinical symptoms . Symptoms vary depending on the level of infection and immune state of the host. They can include diarrhea, cramping, muscle aches, and fever. This disease often leads to meningitis and septicemia in the elderly, and miscarriages in pregnant women . Out of diagnostically confirmed cases of Listeriosis identified during pregnancy, 33.3 percent resulted in the death of the fetus . Listeriosis infects around 1,600 people every year, causing approximately 260 deaths .
The researchers in this study sought an understanding of and some control over this bacteria’s lysozyme-related virulence factor . The main L. monocytogenes strain used was 10403S , which is resistant to streptomycin and was first isolated in 1968 from a skin lesion . They created the following mutants: ΔoatA; ΔpgdA; ΔoatA/ΔpgdA; ΔoatA/Δrli31; ΔpgdA/Δrli31; and ΔoatA/ΔpgdA/Δrli31 to test how necessary each set of genes is in protecting the bacteria from lysozyme susceptibility .
The Enzyme: Lysozyme
Lysozyme is a three-dimensional, polar  enzyme produced by the innate immune system. This defense mechanism protects an organism by dismantling the peptidoglycan that composes a bacterial cell wall, thereby rendering the intruder harmless to the host. . It breaks apart the bacterial cell wall by separating the peptidoglycan at the N-acetylglucosamine and N-acetylmuramic acid bonds . Lysozyme is commonly found in tears, blood, saliva and phagocytic white blood cells such as macrophages .
PgdA, found in L. monocytogenes, as well as Streptococcus pneumoniae, (S. pneumoniae) is a genetic sequence that codes for the enzyme peptidoglycanN-acetylglucosamine deacetylaseA, for which it was named. The 52,699.74-dalton protein contains 463 amino acids . PgdA, which is in the genomes of both pathogenic and nonpathogenic bacteria, is only used in pathogens, adding to their virulence. When active, it modifies the bacterial peptidoglycan to make it chemically unresponsive when contacted by lysozyme .
OatA is the abbreviation for a gene called O-acetyltransferase . This enzyme alters the bacterial cell’s peptidoglycan, subsequently helping it evade damage from lysozyme. Like pgdA, oatA is found in both pathogens and nonpathogens, but is active only in pathogenic bacteria such as L. monocytogenes.
Rli31, if not present, hinders the bacteria from surviving lysozyme in macrophage vacuoles, thereby preventing extensive infections. When injected into mice, strains missing rli31 did not do damage or kill the mice to the extent of the wild type .
In many cases, including in some known pathogens such as S. pneumonia, the ability to resist the effects of lysozyme renders a bacteria pathogenic to a particular host . Wild type L. monocytogenes is lysozyme resistant, but some mutant strains of L. monocytogenes are destroyed by the enzyme. This study solidified the knowledge that mutant L. monocytogenes clones are non-pathogenic because they are adversely affected by lysozyme, while addressing the reasons why this happens. This difference between mutant and wild L. monocytogenes gave the research team a genetic point of comparison, so they looked for differences between the two L. monocytogenes strains .
The research team hypothesized that the presence of pgdA and OatA lead to lysozyme resistance, but found out there is much more to the process. In fact, these genes are present in numerous non-virulent species. The key to activation of this genetic sequence is its connection to DegU transcription factor and the Rli31 noncoding RNA sequence. These two genetic sequences can hinder expression of pgdA. This can adversely affect the bacteria’s resistance to the damaging effects of lysozyme . Without pgdA, it cannot make the peptidoglycan backbone of the bacterial cell an unsuitable molecule for lysozyme activity .
In vivo experiments used laboratory mice infected orally and, in a separate case, intravenously. In both instances, pgdA mutants were unable to cause sustained infection and subsequent disease and bacteria with both mutations (pgdA and oatA) were even more adversely affected by lysozyme in the mice blood and saliva. The intravenous experiment concluded with incremental post injection assessments for organ infection by recovery and subsequent homogenization of the organs, which were later plated on agar and monitored for L. monocytogenes growth .
The researchers found that DegU and Rli31 also play a major role in L. monocytogenes’ ability to withstand contact with lysozyme. When the wild type L. monocytogenes is compared to the strain missing rli31 in Table S1, high-performance liquid chromatography (HPLC) shows that, in all but one instance, the wild type L. monocytogenes retained peptidoglycan integrity more than the strain missing rli31. The peaks related to extracted cell walls are similar .
The researchers experimented with human lysozyme and poultry lysozyme and found that there are differences between the two. Figures S2, A and B show that human lysozyme had an adverse effect on the growth of L. monocytogenes missing dltA and the strain missing mprF and that the chicken lysozyme had no such effect .
There are 13 mutations that can make lysozyme detrimental to L. monocytogenes. Of those 13, five were killed by CAMPs and four by antibiotics. The bacteria were plated with a 1mg lysozyme disc to identify zones of inhibition. When assessed, researchers found a marked difference between the wild type L. monocytogenes and the mutant strains. The zones of inhibition confirmed other associated experiment results, again revealing that mutant strains without pgdA and oatA genetic coding ability were dismantled by lysozyme .
Table S2 shows the 27 strains of L. monocytogenes used in the study . I am inclined to believe in the validity of this research because the group worked with so many strains, put in and took out genes to determine if inclusion or deletion made a difference and because they repeated several of the experimental steps numerous times to ensure the data’s accuracy. My search of associated scientific literature also indicates that similar research is yielding like results, which also lends to the credibility of this paper and its associated conclusions.
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