*M38: Environmental Control of Virulence Gene Expression Flashcards
Regulation of Bacterial Virulence Gene Expression:
- Virulence is typically multifactorial, so it makes “pathogenic sense” for the expression of all necessary virulence genes to be turned on/off at the same time, this is called coordinate regulation.
- Sometimes this coordinate regulation is achieved with a single regulatory element, called a global regulator; in other cases, coordinate regulation is more complicated and involves multiple regulatory elements.
- Environmental changes known to signal changes in virulence gene expression include, temperature, pH, nutrient changes (e.g., iron), and osmolarity. These may act together in vivo.
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Regulation of Bacterial Virulence Gene Expression:
- Usually environmental changes signal the bacterium to coordinately turn on/off virulence gene expression.
a. Sometimes these virulence gene expression changes occur when the pathogen senses macroenvironmental changes, i.e., the bacterium is inside/outside the host.
b. Sometimes these virulence gene expression changes occur when the pathogen senses microenvironmental changes, i.e., the bacterium senses it is inside/outside a host cell. - How do bacteria sense these environmental changes? Two sensing mechanisms, i.e., two component regulatory systems and quorum sensing, are particularly important.
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Two Component Regulatory Systems:
- Two component regulatory systems regulate many bacterial functions, including virulence factor expression.
- Two component regulatory systems are found in both gram-positive and gram-negative bacteria.
- The components of two component regulatory systems include:
a. sensor protein: a transmembrane protein that, when stimulated by the proper environmental signal, undergoes a conformation change, activating the cytoplasmic histidine kinase domain.
b. transcriptional regulator: the activated sensor protein phosphorylates the transcriptional regulator, which is a cytoplasmic protein.
Once phosphorylated, the transcriptional regulator activates expression of some genes, but represses expression of other genes.
c. A single bacterial cell can carry more than one type of two component regulatory system; these different systems can cross-talk to fine-regulate expression of a gene.
i) It is also possible for a two component regulatory system to regulate the activity of another two component regulatory system.
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The PhoP/PhoQ System in Salmonella:
- PhoP is the transcriptional regulator, while PhoQ is the membrane sensor.
- PhoP/PhoQ appears to be essential for virulence.
- Activation of PhoP/PhoQ increases expression of some genes (called pag genes), but represses expression of other genes (called prg genes).
- PhoP/PhoQ mutants do not survive in phagocytes, suggesting that this system is important for survival of Salmonella inside phagocytes.
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The PhoP/PhoQ System in Salmonella:
- PhoP/PhoQ responds to conditions (e.g., low Mg2+ levels) found inside acidified phagosomes, becoming activated when Salmonella enter phagocytes.
a. This activation turns on expression of pag genes needed for intracellular survival, but turns off prg genes that were needed for extracellular survival/early steps in pathogenesis, but which are now unnecessary.
b. Increased expression of pag genes enables Salmonella to survive inside phagocytes by permitting resistance to antimicrobial peptides.
c. One mechanism for this resistance is to activate expression of a second two component regulator (PrmA/PrmB) that, in turn, causes production of enzymes that modify LPS so it no longer binds antimicrobial peptides.
d. Another mechanism for this resistance is to increase production of membrane proteins which somehow protect the Salmonella cell from antimicrobial peptides.
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Quorum Sensing Systems:
- Often bacterial pathogens must reach a critical density in the human body before causing disease.
- Some pathogens sense their population density, and accordingly adjust their virulence factor expression, using quorum sensing.
- Quorum sensing involves production/secretion of a small molecule (a peptide or a homoserine lactone) called an autoinducer.
a. When the bacterial population reaches proper density, sufficient levels of the autoinducer become present to activate transcriptional regulators.
b. the activated transcriptional regulator then leads to increased expression of virulence genes. - An example of a pathogen using quorum sensing is Pseudomonas aeruginosa:
a. Note that activation of a transcriptional regulator leads to production of a new autoinducer, which (in turn) activates transcription of additional virulence genes.
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Cross-Talk Between Quorum-Sensing and Two-Component Regulatory Systems:
- Expression of a virulence gene is often under the control of multiple regulatory systems.
- This can involve cross-talk between quorum-sensing and two component regulatory systems.
- Example: in S. aureus, an autoinducer binds to and activates AgrC, the sensor protein of a two component regulatory system.
- Interestingly, the autoinducer of one S. aureus strain can bind to, but not activate, the AgrC of another S. aureus strain, inhibiting toxin expression by that strain.
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Case Study: A 47 year-old American food worker visits India on a tour. Near the end of her vacation, she becomes ill with a mild fever (38.7°C), abdominal cramps, and constipation. After a few days, she feels better and returns to the USA. A week later she abruptly develops a high fever (40.2°C), diarrhea (six watery stools/day), chills, dark urine and vomiting. Her abdomen is covered with “rose spots”.
She is admitted to the hospital and a blood culture is performed. The culture shows the presence of Gram-negative, lactose-negative rods that are later identified as S. typhi.
Cause: _ spp.
Salmonella spp.
Salmonella spp.:
Biological Characteristics:
a. Nomenclature: many classification schemes have been used for the Salmonellae. For simplicity, we will simply consider two groups of Salmonella, i.e. S. typhi (the primary cause of typhoid fever) and nontyphoid Salmonella (which cause acute gastroenteritis, which sometimes leads to septicemia).
b. Characteristics: Members of the Enterobacteriaceae, these are Gram-negative, facultative anaerobic, motile rods. They do not ferment lactose. Most strains produce H2S (S. typhi makes only trace amounts of H2S).
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Salmonella spp.:
Reservoirs and Transmission:
a. Nontyphoid Salmonella:
i) Reservoir: these bacteria have a zoonotic reservoir, including nearly all common food animals. Pets are also an important reservoir.
ii) Transmission: usually acquired by ingestion of contaminated food (there are probably several million cases/year in USA). Can also be acquired by drinking contaminated water or person-to-person spread by fecal-oral route. Some people can be asymptomatically infected or become carriers (an important reservoir?).
b. S. typhi:
i) Reservoir: Human reservoir only. Human carriers are an important reservoir.
ii) Transmission: spread by fecal-oral contamination.
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Salmonella spp.:
Pathogenesis of Nontyphoid Salmonella:
i) After ingestion, some nontyphoid Salmonella enter the small intestines (maybe later large intestine?), where they adhere to the intestinal epithelium.
ii) The nontyphoid Salmonella then invade the intestinal epithelium, using the Inv/Spa type III secretion system. This triggers membrane ruffling, which results in the internalization of the bacterium via macropinocytosis. Remember: Salmonella can invade both M cells and enterocytes.
iii) Once inside an epithelial cell, the nontyphoid Salmonella remain inside endosomes, which gradually translocate to the basal side of the intestinal cell. During this time, the bacterium is adjusting to life inside an endosome and is also replicating.
iv) When the endosome contacts the basal membrane, the nontyphoid Salmonella cell is released into the lamina propria.
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Salmonella spp.:
Pathogenesis of Nontyphoid Salmonella:
v) In the lamina propria, the nontyphoid Salmonella cell is often phagocytosed by macrophages. The entry of this bacterium is sensed by PhoP/PhoQ, which triggers changes in gene expression that facilitates survival in macrophages.
vi) This increased survival involves, at a minimum, LPS modifications and the expression of additional outer membrane proteins which, together, help the bacterium resist antimicrobial peptides.
vii) In the immunocompetent, the presence of nontyphoid Salmonella in macrophages induces a strong inflammatory response. This produces the characteristic fever, cramps, and (sometimes bloody) diarrhea of Salmonella gastroenteritis. These symptoms usually last for about a week before resolving.
viii) In healthy people, nontyphoid Salmonella typically do not spread beyond the lamina propria. However, in immunocompromised people who cannot mount a strong cell-mediated immune response or sickle cell patients, nontyphoid Salmonella often disseminate, producing systemic disease and (possibly) death. These bacteria can cause septicemia and death in AIDS patients or children with sickle cell disease. Also cause osteomyelitis in sickle cell patients.
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Salmonella spp.:
Pathogenesis of Typhoid Salmonella:
i) Pathogenesis is similar to nontyphoid Salmonella up to the point where S. typhi becomes present in the lamina propria.
ii) Once present in the lamina propria, S. typhi quickly disseminates from the intestines; therefore, the initial GI symptoms of typhoid fever are relatively mild (low grade fever, constipation).
iii) During this initial dissemination, S. typhi moves via the blood and/or lymph to reticuloendothelial tissue. In this tissue, the bacteria multiply until they suddenly emerge (1-2 weeks later) into the blood and bile.
iv) This second dissemination event corresponds to the onset of full- blown typhoid fever. The presence of large numbers of S. typhi in the blood/organs can trigger septic shock and high fever.
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Salmonella spp.:
Pathogenesis of Typhoid Salmonella:
v) Large numbers of S. typhi also enter the intestines via bile; since the intestinal immune system had been exposed to these bacteria only 1-2 weeks earlier, a strong inflammatory response is triggered.
vi) This strong intestinal inflammatory response (and typhoid toxin?) results in diarrhea and granuloma/abscess formation that can lead to hemorrhaging and perforation of the intestine. If perforation occurs, this can result in peritonitis.
vii) Without treatment, there is a 10-20% mortality rate for typhoid fever. In the absence of antimicrobial therapy, activated macrophages are important for recovery.
viii) Some people (particularly those with gallstones) become carriers-they can shed viable bacteria in their feces and be a source of new infections.
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Salmonella spp.:
Diagnosis:
a. Lab work is important: selection of lactose-negative bacteria on MacConkey agar and biochemical tests are important.
b. For nontyphoid Salmonella, stool cultures are informative for diagnosing Salmonella gastroenteritis, as they typically remain positive throughout the illness. Epidemiologic characteristics are also informative. For diagnosing cases of septicemia involving nontyphoid Salmonella, blood cultures are important.
c. For typhoid Salmonella, blood cultures usually test positive before stool or urine cultures, so blood cultures are most often used for initial lab diagnosis of typhoid fever. Clinical signs (e.g., abdominal rose spots) are also helpful.
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