Category:Team Moldevort

Figure 1: C57BL/6 mice were infected aerogenically with a low dose (1x10^8) of either WT or ppe18 KO strains of Mtb. At different time points post infection, mice were sacrificed and CFU counts were measured in lung (A), liver (B) and spleen (C). Lower CFU recovered of the ppe18 KO compared to WT in all three tissues.

In Mycobacterium smegmatis mc2 155, strain mc(2)ΔpknA strain was transformed with pNit vector, pNit-PknA or pNit-PknAK42M. When the inducer was absent the mc(2)ΔpknA:pNit-PknA grew robustly which indicated wild type PknA was capable of functional complementation. Fig 6C shows the growth of M. smegmatis when transformed and showed that PknA is significant for cell growth. When the PknA kinase activity was absent, seen in Fig 6C with pNit vextor and kinase inactive PknA(K42M), there was no growth recovery. Fig 6E and F demonstrated that PknA is necessary for growth. Depletion of PknA in the mc(2)ΔpknA strain resulted in compromised growth, show in Fig 2B.

Mycobacterium tuberculosis (strain H37Rv)

Serine/threonine-protein kinase A

Figure 3B: The cell morphologies of Mycobacterium tuberculosis strains H37Rv (wild-type), Rv-pptr-AB (pknA and pknB conditional mutant, pknA-pknB expression was under the regulation of a pristinamycin inducible promoter [pptr]), and Rv-pptr-AB::PknB (electroporated with pCiT-PknB, containing anhydrotetracycline [ATc] inducible PknB) were examined using scanning electron microscopy. H37Rv cultures were grown for 4 days in the absence of any inducer. Rv-pptr-AB cultures were grown either in the presence or absence of pristinamycin for 2 and 4 days. Rv-pptr-AB::PknB cultures were grown in the presence of ATc (in the absence of pristinamycin) for 2 and 4 days. After 4 days of growth, Rv-pptr-AB and Rv-pptr-AB::PknB cells showed signs of substantial cell-cell fusion and cell lysis, relative to the wild-type H37Rv. These results suggest that PknA plays a role in regulating cell morphology in Mycobacterium tuberculosis.

Figure 4B: Graph shows similar cfu counts for the wild type Mycobacterium tuberculosis H37Rv, the complemented (pknA and pknB) strain, and pknB strain at 24 hours. The wild type and complemented strain expressed increased growth at both 4 and 8 weeks while the cfu counts for the pknB strain was below the detection range at both 4 and 8 weeks post infection.

Figure 6C: Shows a growth analysis through absorbance readings over the course of three hours where the pknA strain grew and the pknA mutant showed no growth nor growth recovery. Figure 6D: When plated on ATc (anhydrotetracycline) plates without pristinamycin the complemented strain grew while the pknA mutant and pknB strain did not grow.

Figure 3B and 3C: Streptococcus pyogenes serotype M1(MGAS5005) with mutation in rocA promoter showed increased transcription of nga and sda1 virulence genes

Figure 4A: Which describes the negative regulation of B-galactosidase via the ccpa protein in Streptococcus pneumoniae strain D39 serotype 2. In the control, the deletion of catabolite control protein A (ccpa) resulted in an increase in the transcript levels of bga and pts. In the the BgaAC mutant, a decrease in transcription occurred for both bga and pts.

Figure 5: Showing that the D39 pts promoter expressed a higher affinity for the CcpA protein rather than the second repressor. The BgaAC mutant bacteria with its pts promoter expressed the higher affinity for the second repressor.

Figure 1: MgrA is needed for clumping in S. aureus strain RN4220. Wild type, a deleted mutant and an exogenous expressed mutant were incubated in plasma and fibrinogen. The images show clumping in the wild type and mgrA expressed mutant.

Figure 2: ebh is a surface protein that blocks clumping. It is repressed by mgrA (Fig 5d and 5a). Other proteins are known to block clumping and are impacted by mgrA, one being sasG and sraP (fig 8+9)

Figure 11: Biofilm formation increased in mutants lacking mgrA, with the increase in ebh. This is due to the up-regulation of the protein sasG, which is one of the proteins up-regulated with ebh as a result of the deletion of mgrA. This protein increases intercellular adhesion, meaning clumping decreases but biofilm formation is enhanced (fig 12)

Fig 9(b + d): The protein phosphokinase A (pknA) was detected in a wild strain Mycobacterium tuberculosis H37Rv using the antibody alpha-Thr(P), which picks up the threonine residues 172 and 174 in the pkna activation loop. PknA was not detected in the strain pknA(K42M), a strain where kinases were inactivated. This suggested that kinases (like pknA) were being activated by auto-phosphorylation.

Fig 9e: In a strain in which the transcription level of pknB is under the control of a pristinamycin inducible promoter, the level of pknA is not impacted whether the strain is grown in the presence or absence of the inducer. This is indicative that pknA is most likely activated through auto-phosphorylation and is independent of pknB

Figure 5A: The lungs of the animals infected with the wild type Mycobacterium tuberculosis H37Rv and the complemented (containing pknA and pknB placed at the L5 site on the chromosome) strain showed an immense infection and large granulomas where the deleted pknA mutant showed normal lung observation.

Figure 5B: looked at lesions in the lungs, showing that the wild type infection caused more damage when compared to the complemented strain.

Figure 5c: A histopathological analysis revealed less severe damage in the complemented strain when compared to the wild type strain.

Figure 4. AvrA, S. typhimurium in mouse