Project Summary: Rampant antibiotic resistance is predicted to cause devastating effects on world health over
the next thirty years. The treatment of simple infections is becoming increasingly complicated by bacteria
resistant to known antibiotics. The effects of resistance on healthcare are exacerbated by a lack of novel
antibiotic development. In particular, Gram-negative bacteria are of increasing concern. All Gram-negative
bacteria have an outer membrane (OM), the presence of which complicates the discovery of new antibiotics,
acting as a barrier to therapeutics and rendering many current antibiotics useless. The OM, which is built by
several molecular machines, is essential in Gram-negative bacteria. Each OM biogenesis machine requires at
least one lipoprotein to function properly. Many lipoproteins are virulence factors, and several are essential to
cellular processes. Gram-negative bacteria must, therefore, navigate the challenge of transporting lipoproteins
from the inner membrane (IM) across the aqueous periplasm to the OM. The Lol system transports lipoproteins
to the OM. LolCDE removes lipoproteins from the IM. LolA, a periplasmic chaperone, receives lipoproteins from
LolCDE and transports them across the periplasm where they are inserted into the OM by LolB. Surprisingly, my
lab recently found that lipoproteins can still reach the OM in the absence of LolAB. Functional lipoprotein
transport in the absence of LolAB demonstrates that an alternate route of lipoprotein transport must exist. This
finding challenges the current paradigm of lipoprotein transport, as the Lol system was previously thought to be
the only mechanism by which lipoproteins reach the OM. Therefore, I hypothesize that an alternate lipoprotein
transport pathway delivers lipoproteins to the OM. Using my lab’s ¿lolAB strain of Escherichia coli, I am uniquely
positioned to identify and define alternate routes of lipoprotein transport. I will use biochemical and genetic
assays to test my hypothesis in two aims. In Aim 1, I will characterize the interaction of LolCDE with the alternate
route of lipoprotein transport. Although lipoproteins are still transported to the OM in the absence of the LolAB
pathway, my lab has confirmed that LolCDE is absolutely essential to all pathways of lipoprotein transport. I will
mutate residues in LolCDE and test the function of the mutants in the alternate lipoprotein transport pathway. I
will then test drug sensitivity to assess OM permeability in LolC mutants. OM biogenesis machine purification
and membrane fractionation will be used to test the ability of mutants to transport lipoproteins. In Aim 2, I will
identify and characterize genes important to the alternate lipoprotein transport pathway. I will use an unbiased
global screen of transposon mutants to identify these genes. I will then use OM biogenesis machine purification
and membrane fractionation to classify genes important to the alternate pathway. Together, these two aims will
close a gap in our understanding of lipoprotein transport in Gram-negative bacteria. Due to the essentiality of
lipoproteins and their transport to the OM, this research will provide important insight that will support future
discovery of therapeutics targeting OM biogenesis.