pPst 9.5 kb- smallest plasmid Found only in Y. Pestis Encodes for plasminogen activator (pla) Cell surface protease Has coagulase activity evident only at temperatures below 30°C, which initiates blockage in flea and forms fibrin matrix that anchors bacteria to proventiculus of flea At 37°C exhibit fibronolytic activities, lyses fibrin at bite site and allows dissemination of bacterium Definitely plays a role in flea transmission, not sure how important the gene product is in mammalian cells.

pPst Other possible functions of Plasminogen activator Produces excess plasmin that causes ineffective structures between inflammatory cells and fibrin Reduces chemoattractants at the infection site possibly via inhibition of IL-8. Encodes bacteriosin pesticin (pst) and its immunity protein (pim)

pCD1 68-75 kb (depending on the strain) Responsible for encoding anti-host genome Y.Pestis, Y.pseudotuberculosis, and Y. Enterocolitis both possess this plasmid Encodes the Low Calcium Response System (LCRS)- controlled by Temperature and Calcium concentration Low Calcium response V antigen-LcrV Yersinia Outer membrane proteins- Yops Specific Yop chaperones- Syc Yop secretion proteins/Type III secretion system- Ysc This plasmid is largely responsible for encoding an anti-host genome. It is necessary for virulence in Y.Pestis and Y. Psuedotuberculosis. LcrV, Yops…all are factors encoded on this plasmid. Im going to talk about each of these factors and each of their function in detail in the next couple of slides.

pCD1 LcrV/V antigen LcrV protein plays a role in regulating the LCRS It is a protective antigen that is associated with resistance to phagocytosis May play an immunosuppressive role, by inhibiting cytokine production Induces IL-10 production by macrophages this downregulates host’s immune response Mutes inflammatory responses by inhibiting cytokine production

pCD1 Yop proteins Altogether there are 29 Yop proteins Yop effector proteins protect Y.pestis from macrophages by destroying phagocytic signaling capabilities There are 6 Yops which directly or indirectly cause disease: Yop E, Yop H, Yop J, Yop O, Yop M, Yop B and D These yops listed here are termed effectors. Yop E through depolymerizing actin microfilaments causes the collapse of the cytoskeleton.

How Yops Thwart Host Immune System ■ Yop E ■ Indirectly depolymerizes actin microfilaments ■ Yop H ■ a protein tyrosine phosphatase capable of dephosphorylating host proteins such as p125FAK and p130Ca at focal adhesions, inhibiting signal transduction necessary for phagocytosis ■ Yop J ■ Binds to MAP kinase family-downregulating TNF-

More Yops Yop O/YpkA Yop M Yop B and D Ser/Thr kinase that probably interferes with signaling pathways Yop M Prevents thrombin platelet aggregation Mutes inflammatory response by sequestering thrombin Yop B and D Translocate effectors across cell membrane

Yops Regulation Temperature and Ca2+ When placed in 37°C and in a medium deprived of Ca2+, Y. Pestis ceases growth and expression of Yops is induced. Yops expression and secretion is induced in Ca2+-containing media by a local signal that occurs at the site of contact between the pathogen and the eukaryotic cell. This leads to the polarized transfer of at least five Yops into the target cell.

pCD1 Syc proteins Small Yop binding proteins Not all Yops are syc associated Act as chaperones by preventing Yop degradation Act as “secretion pilots” leading the Yops to their location of secretion

pCD1 Ysc/ Type III secretion system Yops is mediated by Type III secretion system called Ysc Ysc Type III secretion system consists of: the core Ysc mechanism for secretion through the two bacterial membranes a delivery apparatus (YopB, YopD, YopK, LcrV) control elements (LcrE also called YopN and TyeA at the surface and LcrG in the cytosol) anti-host effector proteins (YopE, YopH, YopM, YpkA and YopJ).

Ysc/Type III secretion system Model for Ysc-dependent and independent secretion of LcrV. LcrV, YopB, YopD, and possibly other proteins (X) are secreted by contact-activated Ysc secretion channels and assemble into an apparatus that mediates targeting of secreted Yops across the plasma membrane and into the cytoplasm of eukaryotic cells. LcrV shed from these structures diffuses into the surrounding medium. The LcrV-transporting contact-activated translocator (VCAT) can also secrete LcrV from Yersinia. Upon contact with the plasma membrane, LcrV not involved in secretion or targeting of Yops is secreted across the inner membrane (IM), periplasm (PP), and outer membrane (OM) of Y. pestis through the VCAT mechanism composed of non-pCD1-encoded proteins. LcrV secreted in this manner is delivered directly to associated eukaryotic cells. [Return to Article] J. Bacteriol.J. Virol.Eukaryot. CellMicrobiol. Mol. Bio. ReviewAll ASM Journals

Yersinia’s Deadly Kiss

All Together Now! Yersiniabaktin=Yersiniabactin (Ybt) Koagulase and Plasminaktivierung= Plasminogen activator Pestizin=Pesticin pTox=pFra pCPC=pPst pYV=pCD1 Fra1=F1 Mureintoxin=Murine Toxin Phagozyte=phagocyte

Clinical Aspects Focus on Bubonic Plague Signs and symptoms Complications Differential Diagnosis Diagnosis Treatment Prognosis Prevention Vaccine Development

Presenting Plague Patients %

Signs & Symptoms Within 2-7 days of Infection: ■ Severe malaise or prostration ---75% ■ High fever, headache ---20%-85% ■ Chills --- 40% ■ Vomiting ---25%-49% ■ Altered mentation ---26%-38% ■ Abdominal pain --- 18% ■ Other: bladder distention, apathy, confusion, fright, anxiety, oliguria, anuria, tachycardia, hypotension, leukocytosis Abrupt onset Presenting symptoms include prostration or severe malaise (75%), headache (20%–85%), vomiting (25%–49%), chills (40%), altered mentation (26%–38%), cough (25%), abdominal pain (18%), and chest pain (13%). Other manifestations of bubonic plague include bladder distention, apathy, confusion, fright, anxiety, oliguria, and anuria. Tachycardia, hypotension, leukocytosis, and fever are frequently encountered. Prostration = extreme physical exhaustion/weakness Septicemia (intermittent at first, rapidly becomes constant) A femoral bubo (a), the most common site of an erythematous, tender, swollen, lymph node bubo, the inflammatory swelling of one or more lymph nodes, usually in the groin; the confluent mass of nodes, if untreated, may suppurate and drain pus

Signs & Symptoms 6-8 hours following onset of symptoms: ■ Pain/tenderness at regional lymph nodes enlarge to be called buboes - extremely painful - occur in groin*, axilla or cervical areas - usually occur in 1 region, but multiple can be seen - may drain pus if left untreated ■ ulcer or skin lesions at site of flea bite <10% of cases

Complications ■ 2 Septicemic Plague due to hematogenous spread (23% of patients) - Purpuric Lesions - Acral Necrosis - DIC disseminated intravascular coagulation - Convulsions - Shock Bubonic plague invades bloodstream… patient develops secondary septicemic plague Sepsis Syndrome with Multi Organ Involvement DIC: tissue necrosis and bleeding following uncontrolled activation of clotting factors & fibrinolytic enzymes Death due to endotoxic shock Purpuric lesions diffuse, hemorrhagic changes in skin (darkened skin changes ="black death”) Acral necrosis

Complications ■ 2 Pneumonic Plague (5-15% of patients) - begins as interstitial pneumonitis;  bacteria in interstitium - show signs and symptoms prior to developing advanced pneumonitis: High fever Severe bronchopneumonia Chest pain Coughing or spitting up blood Difficulty breathing ■ Infection of Buboes ■ Meningitis (< 5%) ■ Death

Distinguishing 1 from 2 Pneumonic Plague ■ no buboes ■ 1-3 day incubation period ■ infectious pneumonitis from onset of symptoms ■ sputum production common ■ less severe evidence of disease in organs other than lungs ■  bacteria in alveoli ■ tracheal & bronchial mucosal hemorrhages ■ can lead to 2 Septicemic Plague symptoms Sudden onset of fever, chills, headache, body pains, weakness, chest discomfort & tightness; Coughing with sputum production; Difficulty breathing; Coughing or spitting up blood; GI symptoms; Pharyngitis - direct inhalation of Y. Pestis

Distinguishing 1 from 2 Septicemic Plague ■ no buboes ■ 1-4 day incubation period ■ more GI symptoms ■ can also lead to 2 Pneumonic Plague (25%) ■ meningitis is 4x more common symptoms Fever and chills Extreme exhaustion Bleeding disorder Necrosis of small vessels Hemorrhagic Skin lesions (purpuric lesions, acral necrosis) Gangrene of extremities (nose or digits) DIC Convulsions Shock - direct bloodstream inoculation with Y. Pestis

Differential Diagnosis Bubonic Plague ■ Chancroid ■ Primary genital herpes ■ 1 or 2 syphilis ■ Strangulated inguinal hernias ■ Streptococcal or staphylococcal adenitis ■ Tularemia ■ Cat scratch disease ■ Mycobacterial infection ■ Lymphogranuloma venereum